Substituted benzyloxypyrimidines and their inactivation of O6 -alkylguanine-DNA alkyltransferase

ABSTRACT

The present invention provides certain novel nitro or nitroso substituted benzyloxy pyrimidines useful as AGT inactivators. An example of such a pyrimidine is a compound of the formula ##STR1## wherein R 1 , is NO 2  or NO, and R 2  is hydrogen, halo, C 1  -C 4  alkyl, C 1  -C 4  hydroxyalkyl, thiol, C 1  -C 4  alkythio, trifluoromethoxy, oxymethanesulfonyl, oxytrifluoromethanesulfonyl, or C 1  -C 4  oxyacyl. The present invention further provides pharmaceutical compositions comprising these compounds, and a method of enhancing the chemotherapeutic treatment of tumor cells in a mammal with an antineoplastic alkylating agent which causes cytotoxic lesions at the O 6  -position of guanine.

This is a divisional of application(s) Ser. No. 08/283,953, filed onAug. 1, 1994 which is now U.S. Pat. No. 5,525,606.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to substituted O⁶ -benzylguanines and6(4)-benzyloxypyrimidines, pharmaceutical compositions comprising suchcompounds, and methods of using such compounds. The subject compoundsare particularly useful in inactivating the human DNA repair protein O⁶-alkylguanine-DNA alkyltransferase.

BACKGROUND OF THE INVENTION

The inactivation of the human DNA repair protein O⁶ -alkylguanine-DNAalkyltransferase (AGT) by O⁶ -benzylguanine leads to a dramaticenhancement in the cytotoxic response of human tumor cells and tumorxenografts to chemotherapeutic drugs whose mechanism of action involvesmodification of DNA guanine residues at the O⁶ -position (Dolan et al.,Proc. Natl. Acad. Sci. U.S.A., 87, 5368-5372 (1990); Dolan et al.,Cancer Res., 51, 3367-3372 (1991); Dolan et al., Cancer Commun., 2,371-377 (1990); Mitchell et al., Cancer Res., 52, 1171-1175 (1992);Friedman et al., J. Natl. Cancer Inst., 84, 1926-1931 (1992); Felker etal., Cancer Chem. Pharmacol., 32, 471-476 (1993); Dolan et al., CancerChem. Pharmacol., 32, 221-225 (1993); Dolan et al., Biochem. Pharmacol.,46, 285-290 (1993)). The AGT inactivating activity of a large number ofO⁶ -benzylguanine analogs have been compared with the aim of obtaininginformation about the types of substituent groups and the sites at whichthey could be attached to O⁶ -benzylguanine without significantlylowering its AGT-inactivating activity (Moschel et al., J. Med. Chem.,35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347 (1994)).While these studies led to the production of a variety of analogs thatwere as potent or somewhat less potent than O⁶ -benzylguanine, none ofthe analogs were better than O⁶ -benzylguanine.

Thus, there remains a need for additional compounds which are capable ofenhancing the chemotherapeutic treatment of tumor cells in a mammal withan antineoplastic alkylating agent which causes cytotoxic lesions at theO⁶ -position of guanine. The present invention provides such compoundsand associated pharmaceutical compositions and treatment methods. Theseand other objects and advantages of the present invention, as well asadditional inventive features, will be apparent from the description ofthe invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The present invention provides 8-substituted O⁶ -benzylguaninederivatives and 4(6)-substituted2-amino-5-nitro-6(4)-benzyloxypyrimidine and2-amino-5-nitroso-6(4)-benzyloxypyrimidine derivatives which have beenfound to be effective AGT inactivators, as well as pharmaceuticalcompositions comprising such derivatives along with a pharmaceuticallyacceptable carrier. The present invention further provides a method ofenhancing the chemotherapeutic treatment of tumor cells in a mammal withan antineoplastic alkylating agent which causes cytotoxic lesions at theO⁶ -position of guanine, by administering to a mammal an effectiveamount of one of the aforesaid derivatives,2,4-diamino-6-benzyloxy-s-triazine, 5-substituted2,4-diamino-6-benzyloxypyrimidines, or 8-aza-O⁶ -benzylguanine, andadministering to the mammal an effective amount of an antineoplasticalkylating agent which causes cytotoxic lesions at the O⁶ -position ofguanine.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a compound of the formula ##STR2##wherein R₁ is a substituent selected from the group consisting of amino,hydroxy, C₁ -C₄ alkylamino, C₁ -C₄ dialkylamino, and C₁ -C₄ acylamino(although, as explained in further detail below, other substituents canbe placed at this 2-position), R₂ is a substituent selected from thegroup consisting of hydrogen, C₁ -C₄ alkyl, C₁ -C₄ aminoalkyl, C₁ -C₄hydroxyalkyl, C₁ -C₄ alkylaminoalkyl, C₁ -C₄ dialkylaminoalkyl, C₁ -C₄cyanoalkyl, C₁ -C₄ carbamoylalkyl, C₁ -C₄ pivaloylalkyl, C₁ -C₄carboalkoxyalkyl, ribose, 2'-deoxyribose, the conjugate acid form of aC₁ -C₄ carboxyalkyl, and the carboxylate anion of a C₁ -C₄ carboxyalkylas the sodium salt (although, as explained in further detail below,other substituents can be placed at this N⁹ -position), and R₃ is asubstituent selected from the group consisting of hydrogen, halo, C₁ -C₄alkyl, C₁ -C₄ hydroxyalkyl, thiol, C₁ -C₄ alkylthio,trifluoromethylthio, C₁ -C₄ thioacyl, hydroxy, C₁ -C₄ alkoxy,trifluoromethoxy, oxymethanesulfonyl, oxytrifluoromethanesulfonyl, C₁-C₄ oxyacyl, amino, C₁ -C₄ aminoalkyl, C₁ -C₄ alkylamino, C₁ -C₄dialkylamino, trifluoromethylamino, ditrifluoromethylamino,aminomethanesulfonyl, C₁ -C₄ aminoacyl, aminotrifluoromethylcarbonyl,formylamino, nitro, nitroso, C₁ -C₄ alkyldiazo, C₅ -C₆ aryldiazo,trifluoromethyl, C₁ -C₄ haloalkyl, C₁ -C₄ cyanoalkyl, cyano, C₁ -C₄alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl, phenyl, phenylcarbonyl, C_(l) -C₄acyl, formyl, C₁ -C₄ alkoxymethyl, phenoxymethyl, C₁ -C₄ vinyl, C₁ -C₄ethynyl, and SO_(n) R' wherein n is 0, 1, 2, or 3 and R' is hydrogen, C₁-C₄ alkyl, amino, or phenyl, with the proviso that R₁ is not amino whenboth R₂ and R₃ are hydrogen. Of particular interest are those compoundswherein R₁ is amino, R₂ is hydrogen or C₁ -C₄ alkyl (preferably methyl),and/or R₃ is amino, C₁ -C₄ alkyl (preferably methyl), hydroxy, halo(preferably bromine), nitro, or trifluoromethyl. Also of particularinterest are those compounds wherein R₁ is hydroxy, C₁ -C₄ alkylamino(preferably methylamino), C₁ -C₄ dialkylamino (preferablydimethylamino), or C₁ -C₄ acylamino (preferably acylamino, i.e., CH₃COHN--), R₂ is hydrogen, and/or R₃ is hydrogen or hydroxy.

The present invention also provides a compound of the formula ##STR3##wherein R₁ is NO₂ or NO, and R₂ is a substituent selected from the groupconsisting of hydrogen, halo, C₁ -C₄ alkyl, C₁ -C₄ hydroxyalkyl, thiol,C₁ -C₄ alkylthio, trifluoromethylthio, C₁ -C₄ thioacyl, hydroxy, C₁ -C₄alkoxy, trifluoromethoxy, oxymethanesulfonyl,oxytrifluoromethanesulfonyl, C₁ -C₄ oxyacyl, C₁ -C₄ aminoalkyl, C₁ -C₄alkylamino, C₁ -C₄ dialkylamino, trifluoromethylamino,ditrifluoromethylamino, aminomethanesulfonyl, C₁ -C₄ aminoacyl,aminotrifluoromethylcarbonyl, formylamino, nitro, nitroso, C₁ -C₄alkyldiazo, C₅ -C₆ aryldiazo, trifluoromethyl, halomethyl, C₁ -C₄haloalkyl, cyanomethyl, C₁ -C₄ cyanoalkyl, cyano, C₁ -C₄alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl, phenyl, phenylcarbonyl, C₁ -C₄acyl, formyl, C₁ -C₄ alkoxymethyl, phenoxymethyl, C₁ -C₄ vinyl, C₁ -C₄ethynyl, and SO_(n) R' wherein n is 0, 1, 2, or 3 and R' is hydrogen, C₁-C₄ alkyl, amino, or phenyl. Of particular interest are those compoundswherein R₂ is hydrogen or a C₁ -C₄ alkyl, preferably methyl,particularly when R₁ is NO₂.

The present invention additionally provides treatment methods, which aregenerally administered via pharmaceutical compositions comprising one ormore of the O⁶ -substituted compounds of the present invention. Inparticular, the present invention provides a method of enhancing thechemotherapeutic treatment of tumor cells in a mammal with anantineoplastic alkylating agent that causes cytotoxic lesions at the O⁶-position of guanine, which method comprises administering to a mammalan effective amount of one or more of the aforedescribed presentinventive compounds, and administering to the mammal an effective amountof an antineoplastic alkylating agent that causes cytotoxic lesions atthe O⁶ -position of guanine. The present invention also includes themethod of enhancing the chemotherapeutic treatment of tumor cells in amammal with an antineoplastic alkylating agent that causes cytotoxiclesions at the O⁶ -position of guanine, which method comprises (i)administering to a mammal an effective amount of

(a) a compound of the formula ##STR4## (b) a compound of the formula##STR5## wherein R is a substituent selected from the group consistingof hydrogen, halo, C₁ -C₄ alkyl, C₁ -C₄ hydroxyalkyl, thiol, C₁ -C₄alkylthio, trifluoromethylthio, C₁ -C₄ thioacyl, hydroxy, C₁ -C₄ alkoxy,trifluoromethoxy, oxymethanesulfonyl, oxytrifluoromethanesulfonyl, C₁-C₄ oxyacyl, amino, C₁ -C₄ aminoalkyl, C₁ -C₄ alkylamino, C₁ -C₄dialkylamino, trifluoromethylamino, ditrifluoromethylamino,aminomethanesulfonyl, C₁ -C₄ aminoacyl, aminotrifluoromethylcarbonyl,formylamino, nitro, nitroso, C₁ -C₄ alkyldiazo, C₅ -C₆ aryldiazo,trifluoromethyl, halomethyl, C₁ -C₄ haloalkyl, cyanomethyl, C₁ -C₄cyanoalkyl, cyano, C₁ -C₄ alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl, phenyl,phenylcarbonyl, C₁ -C₄ acyl, formyl, C₁ -C₄ alkoxymethyl, phenoxymethyl,C₁ -C₄ vinyl, C₁ -C₄ ethynyl, and SO_(n) R' wherein n is 0, 1, 2, or 3and R' is hydrogen, C₁ -C₄ alkyl, amino, or phenyl, or

(c) a compound of the formula ##STR6## and (ii) administering to themammal an effective amount of an antineoplastic alkylating agent whichcauses cytotoxic lesions at the O⁶ -position of guanine.

Various substitutions onto the present inventive compounds and thosecompounds useful in the context of the present inventive method arepossible while retaining the effectiveness of those compounds. Inparticular, the N⁹ position of the compounds of Formulas I and III, andthe 2- and/or 4-positions of the compounds of Formulas I-V, can besubstituted with a variety of substituents (which can be used instead ofany existing substituents at those positions). Such substitutionsinclude aryl, a substituted aryl wherein the aryl substituents areselected from the group consisting of C₁ -C₆ alkyl, C₁ -C₆ alkoxy,nitro, halo, a polycyclic aromatic alkyl containing 2-4 aromatic rings,C₂ -C₆ alkenyl, C₂ -C₆ alkynyl, C_(l) -C₆ haloalkyl, C₁ -C₆hydroxyalkyl, C₁ -C₆ halohydroxy alkyl, acyloxy, an acyloxyalkyl whereinthe alkyl is C₁ -C₆, carboxy, the acid or salt form of carboxyalkylwherein the alkyl is C₁ -C₆, carbonyl, carbamoyl, a carbamoylalkylwherein the alkyl is C₁ -C₆, hydrazinocarbonyl, chlorocarbonyl, cyano,C₂ -C₉ cyanoalkyl, C-formyl, a dialkoxymethyl wherein the alkoxy is C₁-C₆, C-acyl, an alkoxy hydroxyalkyl wherein the alkyl and the alkoxy areC₁ -C₆, carboxymethyl thio, a carboalkoxy alkyl wherein the alkoxy andalkyl are C₁ -C₆, a monoalkylamino hydroxylalkyl wherein the alkyl is C₁-C₆, a dialkylamino hydroxyalkyl wherein the alkyl is C₁ -C₆, aminohydroxyalkyl wherein the alkyl is C₁ -C₆, a peptide derived from theβ-lactone of L-serine, or related amino acids, a monosaccharide selectedfrom the group consisting of aldotetroses, aldopentoses and aldohexoses,a polysaccharide selected from the group consisting of sucrose, lactose,maltose and cellobiose, a nucleic acid segment, a steroid selected fromthe group consisting of testosterone, nortestosterone, anddihydrotestosterone, and SO_(n) R' wherein n is 0, 1, 2, or 3 and R' isH, C₁ -C₆ alkyl or aryl.

The benzene ring of the benzyl groups also may be substituted with oneor more suitable substituents such as, for example, hydrogen, halo,nitro, nitroso, aryl, substituted aryl, aralkyl, substituted aralkyl,polycyclic aromatic arylalkyl, alkyl, cycloalkyl, alkenyl, alkynyl,trifluoromethyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, aryloxy,acyloxy, acyloxyalkyl, oxo, amino, monoalkylamino, dialkylamino,hydrazino, hydroxyamino, acylamino, ureido, thioureido, amidino,guanidino, carboxy, carboxyalkyl, alkoxycarbonyl, carbamoyl,hydrazinocarbonyl, chlorocarbonyl, cyano, cyanoalkyl, C-formyl,dialkoxymethyl, C-acyl, cyanato, isocyanato, thiocyanato,isothiocyanato, carboxymethylthio, aminoalkyl, alkylamino,aminocarboxyalkyl, peptides, carbohydrate, polysaccharide, steroid,heterocycle, aromatic heterocycle, nucleic acid derivative, and SO_(n)R₁ wherein n is 0, 1, 2, or 3, and R₁ is hydrogen, alkyl or aryl. Thehydrocarbon substituents, such as alkyl, alkoxy, aminoalkyl, alkylamino,and the like, will typically be C₁ -C₆, more typically C₁ -C₄.

The range of substituents found useful with respect to the identifiedpositions on the compounds of Formulas I-V are generally electronwithdrawing, as noted in applicants' related U.S. patent applicationSer. No. 07/875,438 and hereinbelow. Further information regardinguseful substituents for AGT depleting compounds of the present inventionis also provided in applicants' other related U.S. patent applicationsSer. Nos. 07/805,634, 07/492,468, and 07/616,913. Collectively, thecompounds useful in the context of the present invention are referred toherein as O⁶ -substituted compounds.

Several 2- and/or 8-substituted 6-benzyloxypurines, substituted6(4)-benzyloxypyrimidines, and a 6-benzyloxy-s-triazine were tested fortheir ability to inactivate the human DNA repair protein, O⁶-alkylguanine-DNA alkyltransferase (AGT, alkyltransferase). Two classesof compounds were identified as being significantly better than O⁶-benzylguanine (the prototype low-molecular-weight inactivator) ininactivating AGT in human HT29 colon tumor cell extracts. These were8-substituted O⁶ -benzylguanines bearing electron-withdrawing groups atthe 8-position and 5-substituted 2,4-diamino-6-benzyloxypyrimidinesbearing electron-withdrawing groups at the 5-position. The latterderivatives were also more effective than O⁶ -benzylguanine ininactivating AGT in intact HT29 colon tumor cells. Both types ofcompounds were as effective or more effective than O⁶ -benzylguanine inenhancing cell killing by 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) ofcolon, breast and prostate cancer cells grown in culture. Provided8-substituted O⁶ -benzylguanine derivatives bearing electron-withdrawingsubstituents at the 8-position and 5-substituted2,4-diamino-6-benzyloxypyrimidines bearing electron-withdrawingsubstituents at the 5-position do not exhibit undesirable toxicity, theyshould be superior to O⁶ -benzylguanine as chemotherapeutic adjuvantsfor enhancing the effectiveness of antitumor drugs whose mechanism ofaction involves modification of the O⁶ -position of DNA guanineresidues. The specific compounds surveyed for AGT inactivating activityare illustrated below. ##STR7##

Preparations of the 8-substituted O⁶ -benzylguanine derivatives8-amino-O⁶ -benzylguanine (1a) and O⁶ -benzyl-8-methylguanine (1b) wereaccomplished by treating 2,8-diamino-6-chloropurine and2-amino-6-chloro-8-methylpurine, respectively, with sodium benzyloxidein benzyl alcohol. O⁶ -Benzyl-8-oxoguanine(O⁶-benzyl-7,8-dihydro-8-oxoguanine, 1c) was prepared by reacting1,1'-Carbonyldiimidazole with 2,4,5-triamino-6-benzyloxypyrimidine(Pfleiderer et al., Chem. Ber., 94, 12-18 (1961)). For convenience thecompound is illustrated in the 8-hydroxy tautomeric form although itmost probably exists in solution in the 8-keto form with a hydrogenattached to the 7-nitrogen atom. O⁶ -Benzyl-8-bromoguanine (1d) wasprepared by bromination of O⁶ -benzylguanine. O⁶-Benzyl-8-trifluoromethylguanine (1e) was prepared by reacting2-amino-6-chloro-8-trifluoromethylpurine with sodium benzyloxide inbenzyl alcohol. 8-Aza-O⁶ -benzylguanine (2) was prepared through nitrousacid treatment of 2,4,5-triamino-6-benzyloxypyrimidine. Compound 2 hadbeen prepared previously by another route (Shealy et al., J. Org. Chem.,27, 4518-4523 (1962)).

With respect to the pyrimidine derivatives (3a-f),4-amino-6-benzyloxy-5-nitropyrimidine (3a) was prepared by treating4-amino-6-chloro-5-nitropyrimidine (Boon et al., J. Chem. Soc., 96-102(1951)) with sodium benzyloxide in benzyl alcohol. Derivatives 3b-d wereprepared by the method of Pfleiderer et al. (Chem. Ber., 94, 12-18(1961)). 2,4-Diamino-6-benzyloxy-5-nitropyrimidine (3e) and2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) were prepared previouslyby Kosary et al. (Acta Pharm. Hung., 49, 241-247 (1989)).

The purines, O⁶ -benzylxanthine (4a) and O⁶ -benzyluric acid (4b) wereprepared by nitrous acid deamination of O⁶ -benzylguanine and O⁶-benzyl-8-oxoguanine, respectively. N² -Acetyl-O⁶ -benzyl-8-oxoguanine(N² -acetyl-O⁶ -benzyl-7,8-dihydro-8-oxoguanine)(4d) was preparedthrough acetylation of O⁶ -benzyl-8-oxoguanine (1c). O⁶-Benzyl-2-fluorohypoxanthine (4c) was prepared previously by Robins andRobins (J. Org. Chem., 34, 2160-2163 (1969)). This material was treatedwith methylamine and dimethylamine to produce O⁶ -benzyl-N²-methylguanine (4e) and O⁶ -benzyl-N²,N² -dimethylguanine (4f),respectively.

Compounds 5a (2-amino-4-benzyloxy-5-nitropyrimidine) and 5b(2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine) were prepared bytreating 2-amino-4-chloro-5-nitropyrimidine and2-amino-4-chloro-6-methyl-5-nitropyrimidine (Boon et al., J. Chem. Soc.,96-102 (1951)), respectively, with sodium benzyloxide in benzyl alcohol.Compound 6 (2,4-diamino-6-benzyloxy-s-triazine) was prepared previouslyunder similar conditions (Wakabayashi et al., Nippon Dojo-HiryyogakuZasshi, 41, 193-200 (1970)). O⁶-Benzyl-8-trifluoromethyl-9-methylguanine (7) was prepared by treatingthe anion of 1e with methyl iodide in N,N-dimethylformamide.

The ability of these compounds to inactivate the AGT protein in HT29human colon tumor cell extracts and in intact HT29 cells is summarizedin Table 1. The data represent the dose of compound required to produce50% inactivation in cell-free extracts upon incubation for 30 min or incells upon incubation for 4 hr.

                  TABLE 1                                                         ______________________________________                                        AGT-Inactivating Activity of 6-Benzyloxypurine, 6(4)-                         Benzyloxypyrimidine, and 6-Benzyloxy-s-triazine Derivatives                                         ED.sub.50 (μM).sup.a                                                         In HT29  In                                                                   cell-free                                                                              HT29                                         Compound                extract  cells                                        ______________________________________                                        2,4-diamino-6-benzyloxy-5-nitrosopyrimidine                                                           0.06     0.02                                         (3d)                                                                          2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e)                                                        0.06     0.02                                         8-aza-O.sup.6 -benzylguanine (2)                                                                      0.07     0.06                                         O.sup.6 -benzyl-8-bromoguanine (1d)                                                                   0.08     0.06                                         O.sup.6 -benzylguanine  0.2      0.05                                         O.sup.6 -benzyl-8-methyl-guanine (1b)                                                                 0.3      0.1                                          O.sup.6 -benzyl-8-oxoguanine (1c)                                                                     0.3      0.15                                         2,4,5-triamino-6-benzyloxy-pyrimidine (3c)                                                            0.4      0.3                                          2-amino-4-benzyloxy-6-methyl-5-nitropyrimidine                                                        0.4      0.06                                         (5b)                                                                          2-amino-4-benzyloxy-5-nitropyrimidine (5a)                                                            0.4      0.05                                         8-amino-O.sup.6 -benzyl-guanine (1a)                                                                  0.7      2                                            2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f)                                                        2        0.8                                          2,4-diamino-6-benzyloxy-5-triazine (6)                                                                4        1.0                                          2,4-diamino-6-benzyloxy-pyrimidine (3b)                                                               15       5                                            O.sup.6 -benzyluric acid (4b)                                                                         25       45                                           4-amino-6-benzyloxy-5-nitropyrimidine (3a)                                                            28       8                                            O.sup.6 -benzyl-2-fluoro-hypoxanthine (4c)                                                            48       12                                           O.sup.6 -benzylxanthine (4a)                                                                          60       35                                           N.sup.2 -acetyl-O.sup.6 -benzyl-8-oxo-guanine (4d)                                                    65       11                                           O.sup.6 -benzyl-N.sup.2 -methyl-guanine (4e)                                                          160      60                                           O.sup.6 -benzyl-N.sup.2, N.sup.2 -dimethyl-guanine (4f)                                               200      110                                          ______________________________________                                         .sup.a The effective dose required to produce 50% inactivation in cellfre     extracts upon incubation for 30 min or in cells upon incubation for 4 hr.     The values for O.sup.6benzylguanine are from Moschel et al., J. Med.          Chem., 35, 4486-4491 (1992).                                             

Within these series of compounds, O⁶ -benzyl-N² -methyl- and O⁶-benzyl-N²,N² -dimethylguanine (4e and 4f) were the least active agentsexhibiting ED₅₀ values for inactivation of AGT in HT29 cell extracts of160 and 200 μM, respectively. For comparison, the ED₅₀ value exhibitedby O⁶ -benzylguanine was 0.2 μM (Table 1). The other 2- and/or8-substituted 6-benzyloxypurines, N² -acetyl-O⁶ -benzyl-8-oxoguanine(4d), O⁶ -benzylxanthine (4a), O⁶ -benzyl-2-fluorohypoxanthine (4c) andO⁶ -benzyluric acid (4b), together with the substituted pyrimidines4-amino-6-benzyloxy-5-nitropyrimidine (3a) and2,4-diamino-6-benzyloxypyrimidine (3b), comprised a group ofincreasingly more active AGT inactivating agents exhibiting intermediateED₅₀ values in the range of 65 to 15 μM.2,4-Diamino-6-benzyloxy-s-triazine (6) and2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) were considerably moreactive than 3b indicating that electron-withdrawing groups at the5-position of a 2,4-diamino-6-benzyloxypyrimidine derivative arepositive contributors to efficient AGT inactivation. This is furtheremphasized by the very high activity exhibited by2,4-diamino-6-benzyloxy-5-nitroso- (3d) and2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e), which contain stronglyelectron-withdrawing nitroso and nitro substituents, respectively. Thesetwo derivatives are the most active AGT inactivators tested to date. Theobservation that 2-amino-4-benzyloxy-5-nitropyrimidine (5a) is much moreactive than 3a indicates that a 2-amino group is critical for highactivity for a 6(4)-benzyloxy-5-nitropyrimidine derivative. Anadditional alkyl group at the 4(6)-position (e.g., as in 5b) does notenhance activity significantly over that for 5a although an amino groupat the 4(6)-position significantly enhances activity. Thus, AGTinactivating activity increases substantially over the series5a=5b<3d=3e. With these considerations in mind the activity of2,4,5-triamino-6-benzyloxypyrimidine (3c) seems exceptional and thereasons for its relatively high activity are unclear at present. It isalso significant that pyrimidines 5a and 5b are quite active in cells,which is not totally predicted by their corresponding activity in HT29cell extracts.

All the O⁶ -benzylguanine analogs 1a-d were much more active than thepurines in the series 4a-f and the activity differences among 1a-d alsoreflect enhancements due to introduction of electron withdrawing groups.Thus, activity increased in the series 8-amino-O⁶ -benzylguanine (1a)<O⁶ -benzyl-8-oxoguanine (1c) <O⁶ -benzyl-8-methylguanine (1b) <O⁶-benzyl-8-bromoguanine (1d) <8-aza-O⁶ -benzylguanine (2). Indeed,derivatives 1d and 2 were essentially as active as pyrimidines 3d and 3ein cell-free extracts although 1d and 2 were somewhat less active incells than expected from their activity in cell-free extracts.

The ability of increasing concentrations of 1a-d, 2, and 3c-e to enhancethe killing of human HT29 colon cancer cells, DU-145 prostate cancercells, and MCF-71 breast cancer cells by BCNU (40 μM) is shown in Tables2, 3, and 4, respectively. The data reflect the number of cell coloniesthat result following exposure to AGT inactivator alone or AGTinactivator 2 hr before exposure to BCNU as described in Dolan et al.(Proc. Natl. Acad. Sci., U.S.A., 87, 5368-5372 (1990)). Data for O⁶-benzylguanine are included for comparison. As indicated, at 10 μMconcentrations, all the 8-substituted purines with the exception of 1awere as effective as O⁶ -benzylguanine in enhancing the cytotoxicity ofBCNU (40 μM); such treatment killed essentially all the tumor cells.Treatment of the cells with the modified 8-substituted O⁶ -benzylguaninealone or BCNU alone had no significant effect on cell colony number. Thecomparatively low activity of 1a in all but the breast cancer cells mayreflect its poor transport into other tumor cell types or its rapidmetabolic conversion to an ineffective AGT inactivator. Its ineffectiveenhancement of BCNU cytotoxicity parallels its relatively poor AGTinactivating ability in colon tumor cells (Table 1).

For the pyrimidines tested, 2,4,5-triamino-6-benzyloxypyrimidine (3c)was as effective as the 8-substituted O⁶ -benzylguanine derivatives andO⁶ -benzylguanine itself in enhancing BCNU toxicity although thenitroso- and nitropyrimidine derivatives (3d and 3e) were similarlyeffective at a 4-fold lower dose.

                  TABLE 2                                                         ______________________________________                                        Killing of HT-29 Colon Cancer Cells                                           by BCNU Combined with AGT Inactivators                                                                           Colony                                                     Inactivator        Formation                                                  Concentration                                                                            BCNU    per 1000                                   Inactivator     (μM)    (μM) cells                                      ______________________________________                                        None                       None    435 ± 63                                None                       40      442 ± 34                                O.sup.6 -benzylguanine                                                                        10         None    431 ± 33                                                10         40      13 ± 6                                                  2.5        40       38 ± 15                                                1          40      277 ± 25                                8-aza-O.sup.6 -benzylguanine (2)                                                              10         None    537 ± 48                                                10         40       2 ± 1                                                  1          40      423 ± 42                                O.sup.6 -benzyl-8-bromoguanine (1d)                                                           10         None    401 ± 22                                                10         40       1 ± 0                                                  1          40      299 ± 30                                O.sup.6 -benzyl-8-oxoguanine (1c)                                                             10         None    401 ± 22                                                10         40      <1                                                         1          40      221 ± 15                                O.sup.6 -benzyl-8-methylguanine (1b)                                                          10         None    513 ± 76                                                10         40      <1                                                         1          40      230 ± 51                                O.sup.6 -benzyl-8-aminoguanine (1a)                                                           10         None    504 ± 30                                                10         40      430 ± 41                                                1          40      475 ± 26                                2,4,5-triamino-6-                                                                             10         None    453 ± 59                                benzyloxypyrimidine (3c)                                                                      10         40       3 ± 1                                                  1          40      487 ± 32                                2,4-diamino-6-benzyloxy-5-                                                                    2.5        None    528 ± 64                                nitrosopyrimidine (3d)                                                                        2.5        40      <1                                                         1          40      19 ± 4                                  214-diamino-6-benzyloxy-5-                                                                    2.5        None    438 ± 25                                nitropyrimidine (3e)                                                                          2.5        40      <1                                                         1          40      45 ± 4                                  ______________________________________                                    

    ______________________________________                                        Killing of DU-145 Prostate Cancer Cells by BCNU Combined                      with AGT Inactivators                                                                                            Colony                                                     Inactivator        Formation                                                  Concentration                                                                            BCNU    per 1000                                   Inactivator     (μM)    (μM) cells                                      ______________________________________                                        None                       None    453 ± 81                                None                       40      394 ± 76                                O.sup.6 -benzylguanine                                                                        10         None    462 ± 68                                                10         40      28 ± 5                                                  1          40      299 ± 18                                8-aza-O.sup.6 -benzylguanine (2)                                                              10         None    452 ± 72                                                10         40      28 ± 5                                                  1          40      248 ± 21                                O.sup.6 -benzyl-8-bromoguanine (1d)                                                           10         None    493 ± 90                                                10         40      16 ± 3                                                  1          40      267 ± 39                                O.sup.6 -benzyl-8-oxoguanine (1c)                                                             10         None    379 ± 34                                                10         40      34 ± 3                                                  1          40      329 ± 43                                O.sup.6 -benzyl-8-methylguanine (1b)                                                          10         None    357 ± 43                                                10         40      50 ± 7                                                  1          40       306 ± 157                              O.sup.6 -benzyl-8-aminoguanine (1a)                                                           10         None    380 ± 36                                                10         40      435 ± 70                                                1          40      295 ± 45                                2,4,5-triamino-6-                                                                             10         None     429 ± 101                              benzyloxypyrimidine (3c)                                                                      10         40      57 ± 7                                                  1          40      378 ± 60                                2,4-diamino-6-benzyloxy-5-                                                                    2.5        None    403 ± 35                                nitrosopyrimidine (3d)                                                                        2.5        40       7 ± 3                                                  1          40      25 ± 4                                                  0.25       40      192 ± 17                                2,4-diamino-6-benzyloxy-5-                                                                    2.5        None    407 ± 80                                nitropyrimidine (3e)                                                                          2.5        40       9 ± 2                                                  1          40      59 ± 6                                                  0.25       40      129 ± 26                                ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Killing of MCF-71 Breast Cancer Cells by BCNU Combined                        with AGT Inactivators                                                                                            Colony                                                     Inactivator        Formation                                                  Concentration                                                                            BCNU    per 1000                                   Inactivator     (μM)    (μM) cells                                      ______________________________________                                        None                       None    426 ± 78                                None                       40      364 ± 60                                O.sup.6 -benzylguanine                                                                        10         None    455 ± 63                                                10         40       4 ± 2                                                  2.5        40      12 ± 6                                  8-aza-O.sup.6 -benzylguanine (2)                                                              10         None    483 ± 27                                                10         40       2 ± 1                                  O.sup.6 -benzyl-8-bromoguanine (1d)                                                           10         None     380 ± 109                                              10         40       3 ± 1                                                  2.5        40       4 ± 3                                  O.sup.6 -benzyl-8-oxoguanine (1c)                                                             10         None    522 ± 78                                                10         40       4 ± 2                                  O.sup.6 -benzyl-8-methylguanine (1b)                                                          10         None    376 ± 76                                                10         40       2 ± 1                                  O.sup.6 -benzyl-8-aminoguanine (1a)                                                           10         None    432 ± 36                                                10         40      95 ± 8                                  2,4,5-triamino-6-                                                                             10         None    448 ± 55                                benzyloxypyrimidine (3c)                                                                      10         40      12 ± 4                                  2,4-diamino-6-benzyloxy-5-                                                                    2.5        None    447 ± 87                                nitrosopyrimidine (3d)                                                                        2.5        40       2 ± 1                                  2,4-diamino-6-benzyloxy-5-                                                                    2.5        None    314 ± 49                                nitropyrimidine (3e)                                                                          2.5        40       2 ± 1                                  ______________________________________                                    

Although the human alkyltransferase is very sensitive to inactivation byO⁶ -benzylguanine and the various compounds described above, a number ofmutants have been generated that are resistant to O⁶ -benzylguanine(Crone and Pegg, Cancer Res., 53, 4750-4753 (1993)). This resistance isprobably caused by a reduction in the space surrounding the active siteof the alkyltransferase, which limits the access to O⁶ -benzylguanine.These mutants are produced by single base changes in thealkyltransferase DNA-coding sequence causing changes in one or two aminoacids in the alkyltransferase (Crone and Pegg, Cancer Res., 53,4750-4753 (1993)). Thus, as indicated in Table 5, changing the prolineresidue at position 140 to alanine (protein P140A) or the glycineresidue at position 156 to an alanine (protein G156A) causes a 20-foldand a 240-fold increase in resistance to O⁶ -benzylguanine,respectively. The alkyltransferase containing an arginine in place of aproline at residue 138 together with an arginine in place of a prolineat residue 140 (protein P138A/P140A) is 88-fold more resistant toinactivation by O⁶ -benzylguanine. It is possible that such resistantmutants will arise or be selected for in tumors under the selectivepressure generated by treatment with O⁶ -benzylguanine plus analkylating agent. More potent inhibitors and/or those of a smaller sizethat are better able to fit into the space of the active site of themutant alkyltransferase can be used to advantage to overcome thisresistance.

                  TABLE 5                                                         ______________________________________                                        Inhibition of Mutant Alkyltransferase Proteins                                by O.sup.6 -Benzylguanine or 2,4-Diamino-6-benzyloxy-5-                       nitrosopyrimidine                                                                      ED.sub.50 (μM).sup.a                                                                      2,4-diamino-6-                                                                benzyloxy-5-                                          Protein    O.sup.6 -benzylguanine                                                                     nitrosopyrimidine                                     ______________________________________                                        Control    0.25         0.05                                                  P140A      5            0.1                                                   P138A/P140A                                                                              22           0.3                                                   G156A      60           1                                                     ______________________________________                                         .sup.a The concentration needed to inactivate 50% of the activity in 30       minutes.                                                                 

As shown in Table 5, 2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (3d)was 50 to 60 times better at inactivating the mutant alkyltransferasesthan O⁶ -benzylguanine. Doses of2,4-diamino-6-benzyloxy-5-nitrosopyrimidine leading to intracellularconcentrations greater than 5 μM will therefore be effective atinactivating such resistant alkyltransferases. Concentrations greaterthan 200 μM of O⁶ -benzylguanine would be needed to get suchinactivation, and these are much more than can be achieved with thiscompound in current formulations. However, 8-substituted O⁶-benzylguanine derivatives that are significantly more potent than O⁶-benzylguanine may be useful in inactivating mutant alkyltransferasesprovided their required intracellular concentrations can be achieved.These data for mutant alkyltransferase inactivation and the datapresented earlier indicate that pyrimidine derivatives bearingelectron-withdrawing groups at the 5-position as well as substituted O⁶-benzylguanine derivatives bearing electron-withdrawing groups at the8-position are superior to O⁶ -benzylguanine for use as adjuvants inchemotherapy with agents whose mechanism of action, like that of BCNU,involves modification of the O⁶ -position of DNA guanine residues.

Other 8-substituted O⁶ -benzylguanine derivatives bearingelectron-withdrawing 8-substituents (e.g., NO₂) are readily available.For example, O⁶ -benzyl-8-nitroguanine could be prepared by treatment of8-nitroguanine (Jones and Robins, J. Am. Chem. Soc., 82, 3773-3779(1960)) with phosphorus oxychloride to produce2-amino-6-chloro-8-nitropurine which when treated with sodiumbenzyloxide in benzyl alcohol would produce the desired O⁶-benzyl-8-nitroguanine.

Additional 2,4-diamino-6-benzyloxypyrimidine derivatives bearingelectron-withdrawing groups other than halogen or nitro groups (e.g.,formyl or cyano groups) could also be readily prepared.2,4-Diamino-5-formyl-6-hydroxypyrimidine, a known compound (Delia andOtteman, Heterocycles, 20, 1805-1809 (1983)), can be treated withphosphorus oxychloride to produce a2,4-diamino-6-chloro-5-formylpyrimidine intermediate, which on treatmentwith sodium benzyloxide in benzyl alcohol produces2,4-diamino-6-benzyloxy-5-formylpyrimidine. Treatment of the formylpyrimidine with hydroxylamine affords2,4-diamino-6-benzyloxy-5-cyanopyrimidine. The preparation of a largenumber of 5-substituted 6(4)-benzyloxypyrimidines or 8-substituted O⁶-benzylguanine derivatives is possible for those skilled in the art ofsynthesis of heterocyclic aromatic compounds (D. J. Brown, "ThePyrimidines," in The Chemistry of Heterocyclic Compounds, Vol. 16, A.Weissberger, Ed., Wiley Interscience, New York, 1962; D. J. Brown, "ThePyrimidines," Supplement I, in The Chemistry of Heterocyclic Compounds,Vol. 16, A. Weissberger and E. C. Taylor, Eds., Wiley Interscience, NewYork, 1970; J. H. Lister, "Fused Pyrimidines Part II Purines," in TheChemistry of Heterocyclic Compounds, Vol. 24 Part II, A. Weissberger andE. C. Taylor, Eds., Wiley Interscience, New York, 1971).

Because many 9-substituted O⁶ -benzylguanine derivatives exhibitexcellent AGT inactivation properties (Moschel et al., J. Med. Chem.,35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347 (1994)),8,9-disubstituted analogs are expected to be similarly active. These canbe readily prepared by reacting the anion of 8-substituted O⁶-benzylguanines (e.g., 1a-e) or the anion of 8-aza-O⁶ -benzylguanine (2)with any of the range of compounds already described (Moschel et al., J.Med. Chem., 35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37,342-347 (1994)) to produce a mixture of isomeric 7,8- and8,9-disubstituted O⁶ -benzylguanine derivatives. The desired8,9-disubstituted derivative can be isolated and purified by silica gelcolumn chromatography as already described (Moschel et al., J. Med.Chem., 35, 4486-4491 (1992); Chae et al., J. Med. Chem., 37, 342-347(1994)). Compound 7 was prepared by treating the anion of compound 1ewith methyl iodide in N,N-dimethylformamide.

The O⁶ -substituted compounds of the present invention can beadministered in any suitable manner to a mammal for the purpose ofenhancing the chemotherapeutic treatment of a particular cancer.Although more than one route can be used to administer a particularcompound, a particular route can provide a more immediate and moreeffective reaction than another route. Accordingly, the describedmethods provided herein are merely exemplary and are in no way limiting.

Generally, the O⁶ -substituted compounds of the present invention asdescribed above will be administered in a pharmaceutical composition toan individual afflicted with a cancer. Those undergoing or about toundergo chemotherapy can be treated with the O⁶ -substituted compoundsseparately or in conjunction with other treatments, as appropriate. Intherapeutic applications, compositions are administered to a patient inan amount sufficient to elicit an effective depression of AGT activitythereby potentiating the cytotoxicity of the aforedescribedchemotherapeutic treatment. An amount adequate to accomplish this isdefined as a "therapeutically effective dose," which is also an "AGTinactivating effective amount." Amounts effective for a therapeutic orprophylactic use will depend on, e.g., the stage and severity of thedisease being treated, the age, weight, and general state of health ofthe patient, and the judgment of the prescribing physician. The size ofthe dose will also be determined by the O⁶ -substituted compoundselected, method of administration, timing and frequency ofadministration as well as the existence, nature, and extent of anyadverse side-effects that might accompany the administration of aparticular O⁶ -substituted compound and the desired physiologicaleffect. It will be appreciated by one of skill in the art that variousdisease states may require prolonged treatment involving multipleadministrations, perhaps using a series of different AGT inactivatorsand/or chemotherapeutic agents in each or various rounds ofadministration.

Suitable chemotherapeutic agents usefully administered in coordinationwith the O⁶ -substituted compounds of the present invention includealkylating agents, such as chloroethylating and methylating agents. Suchagents may be administered using conventional techniques such as thosedescribed in Wasserman et al., Cancer, 36, pp. 1258-1268 (1975), andPhysicians' Desk Reference, 48th ed., Edward R. Barnhart publisher(1994). For example, 1,3-bis(2-chloroethyl)-1-nitrosourea (carmustine orBCNU, Bristol-Myers, Evansville, Ind.) may be administered intravenouslyat a dosage of from about 150 to 200 mg/m² every six weeks. Anotheralkylating agent, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea(lomustine or CCNU, Bristol-Myers), may be administered orally at adosage of about 130 mg/m² every six weeks. Other alkylating agents maybe administered in appropriate dosages via appropriate routes ofadministration known to skilled medical practitioners.

Suitable doses and dosage regimens can be determined by conventionalrange-finding techniques known to those of ordinary skill in the art.Generally, treatment is initiated with smaller dosages that are lessthan the optimum dose of the compound. Thereafter, the dosage isincreased by small increments until the optimum effect under thecircumstances is reached. The present inventive method typically willinvolve the administration of about 0.1 μg to about 50 mg of one or moreof the compounds described above per kg body weight of the individual.For a 70 kg patient, dosages of from about 10 μg to about 200 mg of O⁶-substituted compound would be more commonly used, possibly followed byfurther lesser dosages from about 1 μg to about 1 mg of O⁶ -substitutedcompound over weeks to months, depending on a patient's physiologicalresponse, as determined by measuring cancer-specific antigens or othermeasurable parameters related to the tumor load of a patient.

It must be kept in mind that the compounds and compositions of thepresent invention generally are employed in serious disease states, thatis, life-threatening or potentially life-threatening situations. In suchcases, in view of the minimization of extraneous substances and therelative nontoxic nature of the O⁶ -substituted compounds, it ispossible and may be felt desirable by the treating physician toadminister substantial excesses of these O⁶ -substituted compounds.

Single or multiple administrations of the compounds can be carried outwith dose levels and pattern being selected by the treating physician.In any event, the pharmaceutical formulations should provide a quantityof AGT-inactivating compounds of the invention sufficient to effectivelyenhance the cytotoxic impact of the chemotherapy.

The pharmaceutical compositions for therapeutic treatment are intendedfor parenteral, topical, oral or local administration and generallycomprise a pharmaceutically acceptable carrier and an amount of theactive ingredient sufficient to reduce, and preferably prevent, theactivity of the AGT protein. The carrier may be any of thoseconventionally used and is limited only by chemico-physicalconsiderations, such as solubility and lack of reactivity with thecompound, and by the route of administration.

Examples of pharmaceutically acceptable acid addition salts for use inthe present inventive pharmaceutical composition include those derivedfrom mineral acids, such as hydrochloric, hydrobromic, phosphoric,metaphosphoric, nitric and sulfuric acids, and organic acids, such astartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic,gluconic, succinic, p-toluenesulphonic acids, and arylsulphonic, forexample.

The pharmaceutically acceptable excipients described herein, forexample, vehicles, adjuvants, carriers or diluents, are well-known tothose who are skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone that is chemically inert to the active compounds and one that has nodetrimental side effects or toxicity under the conditions of use. Suchpharmaceutically acceptable excipients preferably include saline (e.g.,0.9% saline), Cremophor EL (which is a derivative of castor oil andethylene oxide available from Sigma Chemical Co., St. Louis, Mo.) (e.g.,5% Cremophor EL/5% ethanol/90% saline, 10% Cremophor EL/90% saline, or50% Cremophor EL/50% ethanol), propylene glycol (e.g., 40% propyleneglycol/10% ethanol/50% water), polyethylene glycol (e.g., 40% PEG400/60% saline), and alcohol (e.g., 40% t-butanol/60% water). The mostpreferred pharmaceutical excipient for use in conjunction with thepresent invention is polyethylene glycol, such as PEG 400, andparticularly a composition comprising 40% PEG 400 and 60% water orsaline.

The choice of excipient will be determined in part by the particular O⁶-substituted compound chosen, as well as by the particular method usedto administer the composition. Accordingly, there is a wide variety ofsuitable formulations of the pharmaceutical composition of the presentinvention.

The following formulations for oral, aerosol, parenteral, subcutaneous,intravenous, intramuscular, interperitoneal, rectal, and vaginaladministration are merely exemplary and are in no way limiting.

The pharmaceutical compositions can be administered parenterally, e.g.,intravenously, subcutaneously, intradermally, or intramuscularly. Thus,the invention provides compositions for parenteral administration thatcomprise a solution of the O⁶ -substituted compound dissolved orsuspended in an acceptable carrier suitable for parenteraladministration, including aqueous and non-aqueous, isotonic sterileinjection solutions.

Overall, the requirements for effective pharmaceutical carriers forparenteral compositions are well known to those of ordinary skill in theart. See Pharmaceutics and Pharmacy Practice, J. B. Lippincott Company,Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250 (1982), andASHP Handbook on Injectable Drugs, Toissel, 4th ed., pages 622-630(1986). Such solutions can contain anti-oxidants, buffers,bacteriostats, and solutes that render the formulation isotonic with theblood of the intended recipient, and aqueous and non-aqueous sterilesuspensions that can include suspending agents, solubilizers, thickeningagents, stabilizers, and preservatives. The compound may be administeredin a physiologically acceptable diluent in a pharmaceutical carrier,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol, isopropanol, or hexadecyl alcohol, glycols, such as propyleneglycol or polyethylene glycol, dimethylsulfoxide, glycerol ketals, suchas 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, such aspoly(ethyleneglycol) 400, an oil, a fatty acid, a fatty acid ester orglyceride, or an acetylated fatty acid glyceride with or without theaddition of a pharmaceutically acceptable surfactant, such as a soap ora detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils useful in parenteral formulations include petroleum, animal,vegetable, or synthetic oils. Specific examples of oils useful in suchformulations include peanut, soybean, sesame, cottonseed, corn, olive,petrolatum, and mineral. Suitable fatty acids for use in parenteralformulations include oleic acid, stearic acid, and isostearic acid.Ethyl oleate and isopropyl myristate are examples of suitable fatty acidesters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-β-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

The parenteral formulations typically will contain from about 0.5 toabout 25% by weight of the active ingredient in solution. Preservativesand buffers may be used. In order to minimize or eliminate irritation atthe site of injection, such compositions may contain one or morenonionic surfactants having a hydrophile-lipophile balance (HLB) of fromabout 12 to about 17. The quantity of surfactant in such formulationswill typically range from about 5 to about 15% by weight. Suitablesurfactants include polyethylene sorbitan fatty acid esters, such assorbitan monooleate and the high molecular weight adducts of ethyleneoxide with a hydrophobic base, formed by the condensation of propyleneoxide with propylene glycol. The parenteral formulations can bepresented in unit-dose or multi-dose sealed containers, such as ampulesand vials, and can be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid excipient, forexample, water, for injections, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules, and tablets of the kind previously described.

Topical formulations, including those that are useful for transdermaldrug release, are well-known to those of skill in the art and aresuitable in the context of the present invention for application toskin.

Formulations suitable for oral administration require extraconsiderations considering the peptidyl and/or carbohydrate nature ofsome of the O⁶ -substituted compounds of the present invention and thelikely breakdown thereof if such compounds are administered orallywithout protecting them from the digestive secretions of thegastrointestinal tract. Such a formulation can consist of (a) liquidsolutions, such as an effective amount of the compound dissolved indiluents, such as water, saline, or orange juice; (b) capsules, sachets,tablets, lozenges, and troches, each containing a predetermined amountof the active ingredient, as solids or granules; (c) powders; (d)suspensions in an appropriate liquid; and (e) suitable emulsions. Liquidformulations may include diluents, such as water and alcohols, forexample, ethanol, benzyl alcohol, and the polyethylene alcohols, eitherwith or without the addition of a pharmaceutically acceptablesurfactant, suspending agent, or emulsifying agent. Capsule forms can beof the ordinary hard- or soft-shelled gelatin type containing, forexample, surfactants, lubricants, and inert fillers, such as lactose,sucrose, calcium phosphate, and corn starch. Tablet forms can includeone or more of lactose, sucrose, mannitol, corn starch, potato starch,alginic acid, microcrystalline cellulose, acacia, gelatin, guar gum,colloidal silicon dioxide, croscarmellose sodium, talc, magnesiumstearate, calcium stearate, zinc stearate, stearic acid, and otherexcipients, colorants, diluents, buffering agents, disintegratingagents, moistening agents, preservatives, flavoring agents, andpharmacologically compatible excipients. Lozenge forms can comprise theactive ingredient in a flavor, usually sucrose and acacia or tragacanth,as well as pastilles comprising the active ingredient in an inert base,such as gelatin and glycerin, or sucrose and acacia, emulsions, gels,and the like containing, in addition to the active ingredient, suchexcipients as are known in the art.

The O⁶ -substituted compounds of the present invention, alone or incombination with other suitable components, can be made into aerosolformulations to be administered via inhalation. The compounds arepreferably supplied in finely divided form along with a surfactant andpropellant. Typical percentages of active compound are 0.01%-20% byweight, preferably 1%-10%. The surfactant must, of course, be nontoxic,and preferably soluble in the propellant. Representative of suchsurfactants are the esters or partial esters of fatty acids containingfrom 6 to 22 carbon atoms, such as caproic, octanoic, lauric, palmitic,stearic, linoleic, linolenic, olesteric and oleic acids with analiphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, suchas mixed or natural glycerides may be employed. The surfactant mayconstitute 0.1%-20% by weight of the composition, preferably 0.25-5%.The balance of the composition is ordinarily propellant. A carrier canalso be included as desired, e.g., lecithin for intranasal delivery.These aerosol formulations can be placed into acceptable pressurizedpropellants, such as dichlorodifluoromethane, propane, nitrogen, and thelike. They also may be formulated as pharmaceuticals for non-pressuredpreparations, such as in a nebulizer or an atomizer. Such sprayformulations may be used to spray mucosa.

Additionally, the compounds and polymers useful in the present inventivemethods may be made into suppositories by mixing with a variety ofbases, such as emulsifying bases or water-soluble bases. Formulationssuitable for vaginal administration may be presented as pessaries,tampons, creams, gels, pastes, foams, or spray formulas containing, inaddition to the active ingredient, such carriers as are known in the artto be appropriate.

The concentration of the O⁶ -substituted compounds of the presentinvention in the pharmaceutical formulations can vary widely, i.e., fromless than about 1%, usually at or at least about 10%, to as much as 20to 50% or more by weight, and will be selected primarily by fluidvolumes, viscosities, etc., in accordance with the particular mode ofadministration selected.

Thus, a typical pharmaceutical composition for intravenous infusioncould be made up to contain 250 ml of sterile Ringer's solution, and 100mg of the O⁶ -substituted compound. Actual methods for preparingparenterally administrable compounds will be known or apparent to thoseskilled in the art and are described in more detail in, for example,Remington's Pharmaceutical Science (17th ed., Mack Publishing Company,Easton, Pa., 1985).

It will be appreciated by one of ordinary skill in the art that, inaddition to the aforedescribed pharmaceutical compositions, the O⁶-substituted compounds of the present inventive method may be formulatedas inclusion complexes, such as cyclodextrin inclusion complexes, orliposomes. Liposomes serve to target the compounds to a particulartissue, such as lymphoid tissue or cancerous hepatic cells. Liposomescan also be used to increase the half-life of the O⁶ -substitutedcompound. Liposomes useful in the present invention include emulsions,foams, micelles, insoluble monolayers, liquid crystals, phospholipiddispersions, lamellar layers and the like. In these preparations, the O⁶-substituted compound to be delivered is incorporated as part of aliposome, alone or in conjunction with a suitable chemotherapeuticagent. Thus, liposomes filled with a desired O⁶ -substituted compound ofthe invention can be directed to the site of a specific tissue type,hepatic cells, for example, where the liposomes then deliver theselected chemotherapeutic-enhancement compositions. Liposomes for use inthe invention are formed from standard vesicle-forming lipids, whichgenerally include neutral and negatively charged phospholipids and asterol, such as cholesterol. The selection of lipids is generally guidedby consideration of, for example, liposome size and stability of theliposomes in the blood stream. A variety of methods are available forpreparing liposomes, as described in, for example, Szoka et al., Ann.Rev. Biophys. Bioeng., 9, 467 (1980), and U.S. Pat. Nos. 4,235,871,4,501,728, 4,837,028, and 5,019,369. For targeting to the cells of aparticular tissue type, a ligand to be incorporated into the liposomecan include, for example, antibodies or fragments thereof specific forcell surface determinants of the targeted tissue type. A liposomesuspension containing an O⁶ -substituted compound may be administeredintravenously, locally, topically, etc. in a dose that varies accordingto the mode of administration, the O⁶ -substituted compound beingdelivered, the stage of disease being treated, etc.

While the efficacy of the O⁶ -substituted compounds of the presentinvention has been demonstrated with respect to particular types ofcancerous cells, e.g., colon, prostate, and breast cancer cells, thepresent invention has applicability to the treatment of any type ofcancer capable of being treated with an antineoplastic alkylating agentwhich causes cytotoxic lesions at the O⁶ -position of guanine. Suchcancers include, for example, colon tumors, prostrate tumors, braintumors, lymphomas, leukemias, breast tumors, ovarian tumors, lungtumors, Wilms' tumor, rhabdomyosarcoma, multiplemyeloma, stomach tumors,soft-tissue sarcomas, Hodgkin's disease, and non-Hodgkin's lymphomas.

Similarly, in view of the mode of action of the O⁶ -substitutedcompounds of the present invention, such compounds can be used inconjunction with any type of antineoplastic alkylating agent whichcauses cytotoxic lesions at the O⁶ -position of guanine. Suchantineoplastic alkylating agents include, for example, chloroethylatingagents (e.g. chloroethylnitrosoureas and chloroethyltriazines) andmonofunctional alkylating agents such as Streptozotocin, Procarbazine,Dacarbazine, and Temozolomide.

Among the chloroethylating agents, the most frequently usedchemotherapeutic drugs are 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea(CCNU, lomustine), 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU,carmustine), 1-(2-chloroethyl)-3-(4-methylcyclohexyl)-1-nitrosourea(MeCCNU, semustine), and1-(2-chloroethyl)-3-(4-amino-2-methyl-5-pyrimidinyl)methyl-1-nitrosourea(ACNU). These agents have been used clinically against tumors of thecentral nervous system, multiple myeloma, melanoma, lymphoma,gastrointestinal tumors, and other solid tumors (Colvin and Chabner,Alkylating Agents. In: Cancer Chemotherapy: Principles and Practice,Chabner and Collins, eds., Lippincott, Philadelphia, pp. 276-313 (1990);McCormick and McElhinney, Eur. J. Cancer, 26, 207-221 (1990)).Chloroethylating agents currently under development with fewer sideeffects are 1-(2-chloroethyl)-3-(2-hydroxyethyl)-1-nitrosourea (HECNU),2-chloroethylmethylsulfonylmethanesulfonate (Clomesone), and 1-N-(2-chloroethyl)-N-nitrosoureido!ethylphosphonic acid diethyl ester(Fotemustine) (Colvin and Chabner, Alkylating Agents. In: CancerChemotherapy: Principles and Practice, Chabner and Collins, eds.,Lippincott, Philadelphia, pp. 276-313 (1990); McCormick and McElhinney,Eur. J. Cancer, 26, 207-221 (1990)). Methylating chemotherapeutic agentsinclude Streptozotocin(2-deoxy-2-(3-methyl-3-nitrosoureido)-D-glucopyranose), Procarbazine(N-(1-methylethyl)-4- (2-methylhydrazino)methyl!benzamide), Dacarbazineor DTIC (5-(3,3-dimethyl-1-triazenyl)-1H-imidazole-4-carboxamide), andTemozolomide (8-carbamoyl-3-methylimidazo5,1-d!-1,2,3,5-tetrazine-4-(3H)-one). Temozolomide is active againstmalignant melanomas, brain tumors, and mycosis fungoides. Streptozotocinis effective against pancreatic tumors. Procarbazine is used to treatHodgkin's disease and brain tumors, and DTIC is used in treatment ofmelanoma and lymphomas (Colvin and Cabner, Alkylating Agents. In: CancerChemotherapy: Principles and Practice, Chabner and Collins, eds.,Lippincott, Philadelphia, pp. 276-313 (1990); Longo, Semin. Concol., 17,716-735 (1990)).

The examples set forth below describe the syntheses of theaforedescribed compounds. As regards the methods and materials set forthin these examples, ¹ H-NMR spectra were recorded on a Varian VXR 500Sspectrometer equipped with Sun 2/110 data stations or a Varian XL 200instrument interfaced to an Advanced data system. Samples were dissolvedin DMSO-d₆ with Me₄ Si as an internal standard. EI mass spectra wereobtained on a reversed geometry VG Micromass ZAB-2F spectrometerinterfaced to a VG 2035 data system. Elemental analyses were performedby Galbraith Laboratories, Inc., Knoxville, Tenn.

Most of the reagents and solvents were from Aldrich Chemical Co., Inc.,Milwaukee, Wis. 8-Aza-O⁶ -benzylguanine (2) (Shealy et al., J. Org.Chem., 27, 4518-4523 (1962)), 2,4-diamino-6-benzyloxypyrimidine (3b)(Pfleiderer and Lohrmann, Chem. Ber., 94, 12-18 (1961)),2,4,5-triamino-6-benzyloxypyrimidine (3c) (Pfleiderer and Lohrmann,Chem. Ber., 94, 12-18 (1961)),2,4-diamino-6-benzyloxy-5-nitrosopyrimidine (3d) (Pfleiderer andLohrman, Chem. Ber., 94, 12-18 (1961)),2,4-diamino-6-benzyloxy-5-nitropyrimidine (3e) (Kosary et al., ActaPharm. Hung., 49, 241-247 (1989)),2,4-diamino-6-benzyloxy-5-bromopyrimidine (3f) (Kosary et al., ActaPharm. Hung., 49, 241-247 (1989)), 4-amino-6-benzyloxy-5-nitropyrimidine(3a) and O⁶ benzyl-2-fluorohypoxanthine (4c) (Robins and Robins, J. Org.Chem., 34, 2160-2163 (1969)) were prepared previously. Alternativesynthetic methods are provided below for some of these compoundstogether with spectroscopic data not provided previously. AGTinactivation studies were carried out as described in Moschel et al., J.Med. Chem., 35, 4486-4491 (1992). Cell killing experiments involvingvarious AGT inactivators in combination with BCNU were carried out as inDolan, et al. (Proc. Natl. Acad. Sci. U.S.A., 87, 5368-5372 (1990)).Cells were treated for 2 h with AGT inactivator prior to exposure toBCNU.

EXAMPLE 1 2,8-Diamino-6-chloropurine

A suspension of 8-aminoguanine (Fischer, Z. Physiol. Chem., 60, 69(1909); Beaman et al., in Zorbach and Tipson, Synthetic Procedures inNucleic Acid Chemistry, Vol. 1, pp 41-43, John Wiley & Sons, New York,1968) (3.0 g, 18.1 mmol) in phosphorus oxychloride (90 mL) andN,N-diethylaniline (3 mL) was refluxed for 30 min and the excessphosphorus oxychloride was evaporated under reduced pressure. Ice (20 g)was added slowly to the resulting solution and the pH was adjusted to 6with a concentrated aqueous sodium hydroxide solution. A yellow solidformed and was collected by filtration, washed with water, and dried togive a green solid. Crystallization from water with charcoal treatmentproduced 2,8-diamino-6-chloropurine as a white solid: yield, 2.11 g(63%); mp >275° C. dec.; ¹ H NMR δ6.09 (s, 2H, NH₂, exchange with D₂ O),6.71 (s, 2H, NH₂, exchange with D₂ O); MS (EI) calcd. m/z for C₅ H₅ N₆³⁵ Cl 184.0264, found 184.0266; calcd. m/z C₅ H₅ N₆ ³⁷ Cl 186.0235,found 186.0237.

EXAMPLE 2 8-Amino-O⁶ -benzylguanine (1a)

2,8-Diamino-6-chloropurine (0.9 g, 4.9 mmol) was added to the solutionof sodium (0.22 g, 10 mmol) in benzyl alcohol (9.0 mL). The solution washeated in a 130° C. oil bath for 5 h, and was poured into water (100 mL)with constant stirring for 10 min. Undissolved solid was removed byfiltration and the filtrate was neutralized with glacial acetic acid.The solution was mixed with methanol (100 mL), and half of the aqueousmethanol solution was loaded on a 3×80 cm Sephadex LH-20 column elutedwith methanol/water (1:1) at 1 mL/min. Column eluent was continuouslymonitored at 280 nm and fractions (10 mL) were collected. The remainderof the reaction mixture in MeOH/H₂ O was chromatographed separatelyunder identical conditions. The desired product eluted in fractions100-130. Evaporation of solvent from the pooled fractions 100-130 fromboth chromatographic runs afforded analytically pure 1a: yield, 0.26 g(21%); mp 269°-271° C. dec.; UV (pH 1) λ_(max) 241 nm (ε=0.699×10⁴), 300(1.109×10⁴); (pH 6.9) 250 (sh) (0.447×10⁴), 292 (1.027×10⁴); (pH 13) 255(sh) (0.355×10⁴), 295 (0.932×10⁴); ¹ H NMR δ5.41 (s, 2H, ArCH₂), 5.70(s, 2H, NH₂, exchange with D₂ O), 6.18 (s, 2H, NH₂, exchange with D₂ O),7.25-7.55 (m, 5H, ArH), 11.1 (br s, 1H, NH, exchanges with D₂ O); MS(EI) calcd. m/z for C₁₂ H₁₂ N₆ O 256.1072, found 256.1059; Anal. (C₁₂H₁₂ N₆ O) C, H, N.

EXAMPLE 3 2-Amino-6-chloro-8-methylpurine

A suspension of 8-methylguanine (Daves et al., J. Am. Chem. Soc., 82,2633-2640 (1960)) (1.0 g, 6.1 mmol) in phosphorous oxychloride (30 mL)and N,N-diethylaniline (1 mL) was refluxed for 3 h. The excessphosphorous oxychloride was evaporated under reduced pressure. Theresulting brown oil was dissolved in ice-water and was neutralized witha concentrated aqueous NaOH solution. After evaporation of the solvent,the solid residue was suspended in 70 mL of H₂ O. Undissolved solid wasfiltered off, and the filtrate was loaded on a 3×80 cm Sephadex LH-20column eluted with methanol/water (1:1) at 1 mL/min. Column eluent wascontinuously monitored at 280 nm and fractions (10 mL) were collected.Evaporation of pooled fractions 50-60 produced2-amino-6-chloro-8-methylpurine as a crude solid. Crystallization fromethanol/water with charcoal treatment afforded2-amino-6-chloro-8-methylpurine as a white solid: yield, 0.57 g (51%);mp >265° C. dec.; ¹ H NMR δ2.39 (s, 3H, CH₃), 6.62 (s, 2H, NH₂, exchangewith D₂ O), 12.56 (s, 1H, NH, exchanges with D₂ O); MS (EI) calcd. m/zfor C₆ H₆ N₅ ³⁵ Cl 183.0312, found 183.0309; calcd. m/z for C₆ H₆ N₅ ³⁷Cl 185.0283, found 185.0286.

EXAMPLE 4 O⁶ -Benzyl-8-methylguanine (1b)

Sodium (0.1 g, 4.4 mmol) was stirred in 4.1 mL of benzyl alcohol untilall sodium had reacted. 2-Amino-6-chloro-8-methylpurine (0.41 g, 2.2mmol) was added, and the reaction mixture was heated in a 130° C. oilbath for 5 h. After cooling to room temperature 40 mL of ether was addedto remove excess benzyl alcohol. The sticky precipitate that formed wascollected by filtration and was dissolved in water (50 mL). The pH ofthe yellow solution was adjusted to 5-6 with glacial acetic acid. Thesolution was mixed with methanol (50 mL) and was loaded on a 3×80 cmSephadex LH-20 column eluted with methanol/water (1:1) at 1 mL/min.Column eluent was continuously monitored at 280 nm and fractions (10 mL)were collected. Evaporation of pooled fractions 78-93 affordedanalytically pure 1b: yield, 0.25 g (44%); mp 214-216 IC; UV (pH 1) λmax238 nm (sh) (ε=0.648×10⁴), 290 (1.136×10⁴); (pH 6.9) 242 (0.758×10⁴),284 (0.897×10⁴); (pH 13) 240 (sh) (0.495×10⁴), 286 (0.932×10⁴); ¹ H NMRδ2.33 (s, 3H, CH₃), 5.46 (s, 2H, ArCH₂), 6.17 (s, 2H, NH₂, exchange withD₂ O),7.34-7.51 (m, 5H, ArH), 12.18 (br s, 1H, NH, exchanges with D₂O),; MS (EI) calcd. m/z for C₁₃ H₁₃ N₅ O 255.1120, found 255.1125; Anal.(C₁₃ H₁₃ N₅ O.1/4 H₂ O) C, H, N.

EXAMPLE 5 O⁶ -Benzyl-8-oxoguanine (1c)

2,4,5-Triamino-6-benzyloxypyrimidine (Pfleiderer et al., Chem. Ber., 94,12-18 (1961)) (1.85 g, 8 mmol) and 1,1'-carbonyldiimidazole (1.30 g, 8mmol) were dissolved in anhydrous N,N-dimethylformamide (5 mL) underargon. The solution was stirred at room temperature overnight and wasmixed with water (200 mL) to precipitate a white solid. The solid wascollected by filtration, and dissolved in 250 mL of aqueous 2N NaOHsolution. Undissolved material was removed by filtration, and thefiltrate was neutralized with glacial acetic acid to precipitate a whitesolid. The solid was collected by filtration, was washed with water, andwas recrystallized from 50% aqueous ethanol to afford analytically pure1c: yield, 1.63 g (79%); mp 256°-257° C. dec.; UV (pH 1) λ_(max) 243 nm(ε=0.717×10⁴), 306 (1.499×10⁴); (pH 6.9) 243 (0.915×10⁴), 290(1.108×10⁴); (pH 13) 249 (sh) (0.443×10⁴), 293 (1.368×10⁴); ¹ H NMRδ5.41 (s, 2H, ArCH₂), 6.13 (s, 2H, NH₂, exchange with D₂ O), 7.33-7.51(m, 5H, ArH), 10.46 (s, 1H, exchanges with D₂ O), 11.04 (s, 1H,exchanges with D₂ O); MS (EI) Calcd. m/z for C₁₂ H₁₁ N₅ O₂ : 257.0912.Found: 257.0914. Anal. (C₁₂ H₁₁ N₅ O₂.1/2 H₂ O) C, N, H.

EXAMPLE 6 O⁶ -Benzyl-8-bromoguanine (1d)

Bromine (0.26 mL, 5.1 mmol) was added slowly to the solution of O⁶-benzylguanine (1.205 g, 5.0 mmol) in anhydrous DMF (10 mL) under argon.The resulting deep green solution was stirred at room temperatureovernight. The solution was mixed with water (70 mL) to precipitatecrude product. This product was collected by filtration and wasdissolved in 50% aqueous methanol (100 mL). The solution was loaded on a3×80 cm Sephadex LH-20 column eluted with methanol/water (1:1) at 1mL/min. Column eluent was continuously monitored at 280 nm and fractions(10 mL) were collected. The desired product eluted in fractions 110-190.Evaporation of solvent from the pooled fractions 110-190 afforded 1d asa pale yellow solid. Crystallization from ethanol/water (1:1) producedanalytically pure 1d: yield, 0.166 g (10%); mp 135°-137 ° C. dec.; UV(pH 1) λ_(max) 236 nm (sh) (ε=0.517×10⁴), 294 (1.429×10⁴); (pH 6.9) 244(0.666×10⁴), 287 (1.043×10⁴); (pH 13) 245 (sh) (0.544×10⁴), 289(1.030×10⁴); ¹ H NMR δ5.45 (s, 2H, ArCH₂), 6.35 (s, 2H, NH₂, exchangewith D₂ O), 7.34-7.52 (m, 5H, ArH), 13.08 (b s, 1H, NH, exchanges withD₂ O) ; MS (EI) calcd. m/z for C₁₂ H₁₀ N₅ O⁷⁹ Br 319.0068, found319.0069; calcd. m/z for C₁₂ H₁₀ N₅ O⁸¹ Br 321.0048, found 321.0048;Anal. (C₁₂ H₁₀ N₅ OBr3/2 H₂ O) C, H, N, Br.

EXAMPLE 7 8-Aza-O⁶ -Benzylguanine (2) Glacial acetic acid (1 mL) wasadded into the mixture of 2,4,5-triamino-6-benzyloxypyrimidine (0.231 g,1.0 mmol) and sodium nitrite (0.069 g, 1.0 mmol) in acetone (5 mL). Theresulting mixture was stirred at room temperature for 2 h. The solutionwas poured in water (100 mL) with stirring to precipitate a crude solid.The solid was collected by filtration and air dried. Crystallizationfrom ethanol/water (1:1) with charcoal treatment produced 2 as a whitesolid: yield, 105 mg (43%); mp 191°-192° C. (192°-193° C.; Shealy et.al., J. Org. Chem., 27, 4518-4523 (1962)); ¹ H NMR δ5.56 (s, 2H, ArCH₂),7.00 (s, 2H, NH₂, exchange with D₂ O), 7.41-7.58 (m, 5H, ArH) ; MS (EI)calcd. m/z for C₁₁ H₁₀ N₆ O 242.0916, found 242.0924. EXAMPLE 84-Amino-6-benzyloxy-5-nitropyrimidine (3a)

4-Amino-6-chloro-5-nitropyrimidine (Boon et al., J. Chem. Soc., 96-102(1951)) (1.5 g, 8.6 mmol) was added to a solution of sodium (0.23 g, 9.9mmol) in benzyl alcohol (14 mL). The solution was heated in a 130° C.oil bath for 3.5 h, and was poured into benzene (50 mL). A yellow solidwas collected by filtration and washed with benzene. Crystallizationfrom benzene/ether afforded an analytically pure sample of 3a: yield,0.71 g (34%); mp 149°-150° C.; UV (pH 1) λ_(max) 284 nm (ε=0.368×10⁴),333 (0.488×10⁴); (pH 6.9) 284 (0.329×10⁴), 336 (0.470×10⁴); (pH 13) 290(0.344×10⁴), 333 (0.494×10⁴); ¹ H NMR δ5.50 (s, 2H, ArCH₂), 7.33-7.49(m, 5H, ArH), 8.12-8.24 (br d, 2H, NH_(a) and NH_(b), exchange with D₂O), 8.24 (s, 1H, H-2); MS (EI) calcd. m/z for C₁₁ H₁₀ N₄ O₃ 246.0752,found 246.0751; Anal. (C₁₁ H₁₀ N₄ O₃) C, H, N.

EXAMPLE 9 2,4-Diamino-6-benzyloxy-5-nitropyrimidine (3e)

2,4-Diamino-6-chloro-5-nitropyrimidine (O'Brien et. al., J. Med. Chem.,9, 573-575 (1966)) (1.0 g, 5.28 mmol) was added to a solution of sodium(0.14 g, 6.08 mmol) in benzyl alcohol (9 mL). The solution was heated ina 160° C. oil bath for 3.5 h and the solvent was evaporated underreduced pressure to provide a yellow solid. This solid was washed withwater, and air dried. Crystallization from benzene/ether gave a paleyellow filamentous solid: yield, 0.69 g (50%); mp 194°-195° C. (171° C.;Kosary et. al., Acta. Pharm. Hung., 49, 241-247 (1989)); UV (pH 1)λ_(max) 236 nm (sh) (ε=1.452×10⁴), 264 (0.522×10⁴), 321 (1.294×10⁴); (pH6.9) 242 (sh) (0.965×10⁴), 337 (1.493×10⁴); (pH 13) 242 (sh)(0.952×10⁴), 338 (1.479×10⁴); ¹ H NMR δ5.43 (s, 2H, ArCH₂), 7.26 (br s,2H, NH₂, exchange with D₂ O), 7.33-7.51 (m, 5H, ArH), 7.93 (br s, 2H,NH₂, exchange with D₂ O); MS (EI) calcd. m/z for C₁₁ H₁₁ N₅ O₃ 261.0861,found 261.0866; Anal. (C₁₁ H₁₁ N₅ O₃).

EXAMPLE 10 O⁶ -Benzylxanthine (4a)

A suspension of O⁶ -benzylguanine (0.83 g, 3.4 mmol) in acetone (15 mL)was poured into a solution of sodium nitrite (5 g) in 15 mL of H₂ O.Acetic acid (8 mL) was added to the suspension with stirring. Minimumamounts of acetone were added as necessary to dissolve any suspendedsolid. The resulting pale yellow-green solution was stirred for 3 h. Apale green precipitate that formed was collected by filtration andwashed with water (200 mL). Recrystallization of the air-dried solidfrom ethanol/water (1:1) afforded analytically pure 4a: yield, 0.43 g(52%); mp 145°-147° C. dec.; UV (pH 1) λ_(max) 270 nm (ε=0.749×10⁴); (pH6.9) 286 (1.143×10⁴); (pH 13) 290 (0.914×10⁴); ¹ H NMR δ5.49 (s, 2H,ArCH₂), 7.36-7.54 (m, 5H, ArH), 8.02 (s, 1H, H-8), 11.8 (br s, 1H, NH,exchanges with D₂ O), 13.2 (br s, 1H, NH, exchanges with D₂ O); MS (EI)calcd. m/z for C₁₂ H₁₀ N₄ O₂ 242.0803, found 242.0828; Anal. (C₁₂ H₁₀ N₄O₂ H₂ O) C, H, N.

EXAMPLE 11 O⁶ -Benzyluric acid (4b)

Sodium nitrite (1.5 g, 43 mmol) dissolved in water (5 mL) was added to asuspension of O⁶ -benzyl-8-oxoguanine (1c) (0.257 g, 1.0 mmol) inacetone (5 mL). Glacial acetic acid (3 mL) was added to the suspensionwith stirring. After stirring for 3 h at room temperature a brightyellow precipitate formed. The suspension was mixed with water (150 mL)and undissolved solid was filtered off. Saturated aqueous sodiumcarbonate solution was added to the filtrate to adjust the pH toapproximately 5. A yellow precipitate (130 mg) was collected and washedwith water. This solid was crystallized from 50% aqueous ethanol to givean analytically pure sample of 4b: yield, 75 mg (29%); mp >230° C.; UV(pH 1) λ_(max) 236 nm (sh) (ε=0.972×10⁴), 299 (1.427×10⁴); (pH 6.9) 240(sh) (0.821×10⁴), 304 (2.134×10⁴); (pH 13) 245 (sh) (0.846×10⁴), 297(1.861×10⁴); ¹ H NMR δ5.43 (s, 2H, ArCH₂), 7. 35-7.51 (m, 5H, ArH),10.76 (s, 1H, NH, exchanges with D₂ O), 11.23 (s, 1H, NH, exchanges withD₂ O), 11.39 (s, 1H, NH, exchanges with D₂ O); MS (EI) calcd. m/z forC₁₂ H₁₀ N₄ O₃ 258.0752, found 258.0753; Anal. (C₁₂ H₁₀ N₄ O₃.5/2 H₂ O)C, H, N.

EXAMPLE 12 Diacetyl-O⁶ -benzyl-8-oxoguanine

Acetic anhydride (2 mL) was added to the suspension of O⁶-benzyl-8-oxoguanine (1c) (0.257 g, 1.0 mmol) in dry toluene (10 mL).The suspension was vigorously refluxed for 24 hr, and was cooled to roomtemperature. After storing at 4° C. for 4 hr, the resulting precipitatewas collected by filtration, washed with benzene and air dried to givean analytically pure sample of a diacetylated product: yield, 0.287 g(84%); mp 272°-274° C. dec.; UV (100% MeOH) λ_(max) 275 nm(ε=1.313×10⁴); (pH 1) 275 (1.143×10⁴); (pH 6.9) 238 (0.995×10⁴), 276(1.115×10⁴); (pH 13) 285 (2.138×10⁴); ¹ H NMR δ2.18 (s, 3H, CH₃), 2.57(s, 3H, CH₃), 5.51 (s, 2H, ArCH₂), 7.30-7.57 (m, 5H, ArH), 10.41 (s, 1H,exchanges with D₂ O), 12.30 (s, 1H, exchanges with D₂ O); MS (EI) Calcd.m/z for C₁₆ H₁₅ N₅ O₄ : 341.1123. Found: 341.1130. Anal. (C₁₆ H₁₅ N_(5O)₄) C, N, H.

EXAMPLE 13 N² -Acetyl-O⁶ -benzyl-8-oxoguanine (4d)

Diacetyl-O⁶ -benzyl-8-oxoguanine (85 mg, 0.25 mmol) was dissolved inmethanol (10 mL) and ammonium hydroxide (28%, 5 mL) and was allowedstand for 1 hr. The clear solution became cloudy and a precipitateformed on standing. The precipitate was collected by filtration, washedwith water, and dried to give an analytically pure sample of 4d: yield,48 mg (65%); mp 335°-337° C. dec.; UV (pH 1) λ_(max) 276 nm(ε=1.723×10⁴), 303 (sh) (0.679×10⁴); (pH 6.9) 276 (1.379×10⁴); (pH 13)284 (1.683×10⁴); ¹ H NMR δ2.15 (s, 3H, CH₃), 5.49 (s, 2H, ArCH₂),7.30-7.55 (m, 5H, ArH), 10.21 (s, 1H, exchanges with D₂ O), 10.99 (s,1H, exchanges with D₂ O), 11.60 (s, 1H, exchanges with D₂ O; MS (EI)Calcd. m/z for C₁₄ H₁₃ N₅ O₃ : 299.1018. Found: 299.1023. Anal. (C₁₄ H₁₃N₅ O₃) C, N, H.

EXAMPLE 14 O⁶ -Benzyl-2-fluorohypoxanthine (4c)

O⁶ -Benzylguanine (1.21 g, 5 mmol) was added to 100 mL of 48% fluoboricacid at -20° C.. Sodium nitrite (1.23 g, 35 mmole) was dissolved inwater (5 mL) and 2.5 mL of this sodium nitrite solution was added slowlyto the cold fluoboric acid solution. The resulting mixture was stirredfor 1 h at or below -15° C. Additional fluoboric acid (25 mL) was addedfollowed by an additional 2.5 mL of the aqueous sodium nitrite solution.After stirring for an additional 1 h below -15° C., fluoboric acid (25mL) was again added and stirring was continued for 1 h. The resultingsolution was neutralized with saturated aqueous sodium carbonatesolution at -20° C. and was allowed to warm to room temperature. A whiteprecipitate that formed was collected by filtration and was washed withwater and dried under vacuum to afford crude 4c: yield, 0.52 g, 43%. Ananalytical sample was prepared by chromatography on a Sephadex LH-20column (3×80 cm) eluted with methanol/water (1:1) at 1 mL/min. Thedesired 4c eluted in fractions 66-77: mp 182°-183° C. (184°-185° C.;Robins and Robins, J. Org. Chem., 34, 2160-2163 (1969)); UV (pH 1)λ_(max) 256 nm (ε=1.117×10⁴) ; (pH 6.9) 257 (1.078×10⁴) (pH 13) 264(1.063×10⁴); ¹ H NMR δ5.60 (s, 2H, ArCH₂), 7.37-7.57 (m, 5H, ArH), 8.40(s, 1H, H-8), 13.60 (s, 1H, NH, exchanges with D₂ O), ¹⁹ F NMR δ23.54downfield from trifluoroacetic acid standard; MS (EI) calcd. m/z for C₁₂H₉ FN₄ O 244.0760, found 244.0756; Anal. (C₁₂ H₉ FN₄ O.2/3 H₂ O) C, H,N.

EXAMPLE 15 O⁶ -Benzyl-N² -methylguanine (4e)

Fluoboric acid (48%, 30 mL) was cooled to -20° C. in an dry ice-acetonebath. O⁶ -Benzylguanine (0.362 g, 1.5 mmol) was added with stirring.Sodium nitrite (0.369 g, 10.5 mmol) was dissolved in water (1 mL) and0.5 mL of this solution was added slowly to the cold fluoboric acidsolution. The resulting solution was stirred at or below -15° C. for 1h. More fluoboric acid (5 mL) was then added followed by 0.5 mL of thesodium nitrite solution. After stirring for 1 h at or below -15° C.,fluoboric acid (5 mL) was again added and stirring was continued for anadditional 1 h. Methylamine (40% in water, 60 mL) was then added at -20°C., and the resulting basic solution was stirred at room temperature for2 days. The solvent was evaporated under reduced pressure to produce awhite solid. The solid was suspended in 50 mL of H₂ O with stirring for10 min. Undissolved material was collected by filtration and washed withwater. This solid was dissolved in 40 mL methanol/water (1:1) to whichwas added 1.2 mL of 28% aqueous ammonia solution. The solution wasloaded on a 3×80 cm Sephadex LH-20 column eluted with MeOH/H₂ O/NH₄ OH(30:70:3) at 1 mL/min. Column eluent was continuously monitored at 280nm and fractions (10 mL) were collected. Evaporation of the pooledfractions 106-127 gave an analytically pure sample of 4e: yield, 85 mg(22%); mp 189°-190° C.; UV (pH 1) λ_(max) 238 nm (sh) (ε=0.665×10⁴), 297(0.904×10⁴); (pH 6.9) 246 (0.898×10⁴), 290 (0.676×10⁴); (pH 13) 240 (sh)(0.615×10⁴), 294 (0.674×10⁴); ¹ H NMR δ2.30 (d, 3H, CH₃), 5.50 (s, 2H,ArCH₂), 6.75 (m, 1H, MeNH, exchanges with D₂ O), 7.31-7.53 (m, 5H, ArH),7.82 (s, 1H, H-8), 12.53 (s, 1H, NH, exchanges with D₂ O); MS (EI)calcd. m/z for C₁₃ H₁₃ N₅ O 255.1120, found 255.1107; Anal. (C₁₃ H₁₃ N₅O.1/2 H₂ O) C, H, N.

EXAMPLE 16 0⁶ -Benzyl-N²,N² -dimethylguanine (4f)

Fluoboric acid (48%, 40 mL) was cooled to -20° C. in an dry ice-acetonebath. O⁶ -Benzylguanine (0.482 g, 2.0 mmol) was added with stirring.Sodium nitrite (0.492 g, 14.0 mmol) was dissolved in water (2 mL) and 1mL of this solution was added slowly to the cold fluoboric acidsolution. The resulting solution was stirred at or below -15° C. for 1h. More fluoboric acid (10 mL) was added followed by the addition of 1mL of the sodium nitrite solution. After stirring for 1 h at or below-15° C., additional fluoboric acid (10 mL) was added with stirring for 1h. Dimethylamine (40% in water, 60 mL) was then added to the solution at-20° C., and the resulting mixture was allowed to warm to roomtemperature. The suspension became a clear solution and a precipitateformed within 10 min. After standing overnight at room temperature theprecipitate was collected by filtration and was washed with water. Thesolid was crystallized from 50% aqueous ethanol to give an analyticallypure sample of 4f: yield, 0.25 g (46%); mp 220°-221° C. dec.; UV (pH 1)λ_(max) 248 nm (sh) (ε=0.512×10⁴), 303 (0.908×10⁴); (pH 6.9) 251(1.152×10⁴), 299 (0.686×10⁴); (pH 13) 248 (sh) (0.766×10⁴), 299(0.710×10⁴); ¹ H NMR δ3.12 (s, 6H, CH₃), 5.54 (s, 2H, ArCH₂), 7.36-7.51(m, 5H, ArH), 7.84 (s, 1H, H-8), 12.56 (s, 1H, NH, exchanges with D₂ O);MS (EI) calcd. m/z for C₁₄ H₁₅ N₅ O 269.1276, found 269.1254; Anal. (C₁₄H₁₅ N₅ O) C, H, N.

EXAMPLE 17 2,4-Diamino-6-benzyloxy-5-bromopyrimidine (3f)

2,4-Diamino-5-bromo-6-chloropyrimidine (Phillips et. al., J. Org. Chem.,29, 1488-1490 (1963)) (2.3 g, 10 mmol) was added to a solution of sodium(0.29 g, 12.5 mmol) in benzyl alcohol (10 mL) under argon. The solutionwas heated in a 130° C. oil bath for 3 h and the benzyl alcohol wasevaporated under reduced pressure to give a white solid. This solid waswashed with water, and air dried. Crystallization from 50% aqueousethanol gave white crystalline needles of 3f: yield, 2.32 g (76%); mp165°-166° C. (lit. 136° C.; Kosary et. al., Acta Pharm. Hung., 49,241-247 (1989)); UV (pH 1) λ_(max) 236 nm (ε=0.873×10⁴), 291(1.388×10⁴); (pH 6.9) 236 (0.850×10⁴), 277 (0.835×10⁴); (pH 13) 234(0.869×10⁴), 277 (0.827×10⁴); ¹ H NMR δ5.30 (s, 2H, ArCH₂), 6.15 (s, 2H,NH₂, exchange with D₂ O), 6.32 (s, 2H, NH₂, exchange with D₂ O),7.31-7.45 (m, 5H, ArH); MS (EI) calcd. m/z for C₁₁ H₁₁ N₄ O⁷⁹ Br294.0115, found 294.0127; calcd. m/z for C₁₁ H₁₁ N₄ O⁸¹ Br 296.0094,found 296.0083; Anal. (C₁₁ H₁₁ N₄ OBr) C, H, N.

EXAMPLE 18 2-Amino-4-chloro-5-nitropyrimidine

A suspension of 2-amino-4-hydroxy-5-nitropyrimidine (5.0 g, 32.1 mmol)in phosphorous oxychloride (100 mL) was refluxed overnight, and theexcess phosphorous oxychloride was evaporated under reduced pressure.The residue was mixed with ice (100 g) in an ice-bath, and the mixturewas neutralized with concentrated aqueous sodium carbonate solution. Ayellow precipitate was collected by filtration and washed with water:yield, 1.39 g (25%); mp 191°-194° C. dec.; ¹ H NMR δ8.45 (br s, 2H, NH₂,exchange with D₂ O), 9.03 (s, 1H, H-6); MS (EI) calcd. m/z for C₄ H₃ N₄O₂ ³⁵ Cl 173.9944, found 173.9934; calcd. m/z for C₄ H₃ N_(4O) ₂ ³⁷ Cl175.9915, found 175.9916.

EXAMPLE 19 2-Amino-4-benzyloxy-5-nitropyrimidine (5a)

2-Amino-4-chloro-5-nitropyrimidine (0.70 g, 4.0 mmol) was added to asolution of sodium (0.12 g, 5.2 mmol) in benzyl alcohol (8 mL) underargon. The solution was heated in a 130° C. oil bath for 3 h, andapproximately half of the benzyl alcohol was evaporated under reducedpressure. The residue was poured into water (50 mL) with constantstirring for 10 min. After neutralization with glacial acetic acid, abrown precipitate formed which was collected by filtration and washedwith water. This solid was crystallized from benzene to give 5a as agolden crystalline solid: yield, 126 mg (13%); mp 164°-167° C.; UV(pH 1) λ_(max) 262 nm (ε=0.879×10⁴), 295 (sh) (0.571×10⁴); (pH 6.9) 235(sh) (0.448×10⁴), 273 (0.360×10⁴), 326 (1.085×10⁴); (pH 13) 273(0.404×10⁴), 327 (1.055×10⁴); ¹ H NMR δ5.51 (s, 2H, ArCH₂), 7.35-7.54(m, 5H, ArH), 8.05 (d, 2H, NH₂, exchange with D₂ O), 8.92 (s, 1H, H-6);MS (EI) calcd. m/z for C₁₁ H₁₀ N₄ O₃ 246.0752, found 246.0758; Anal.(C₁₁ H₁₀ N₄ O₃) C, H, N.

EXAMPLE 20 2-Amino-4-benzyloxy-6-methyl-5-nitropyrimidine (5b)

2-Amino-4-chloro-6-methyl-5-nitropyrimidine (Boon et al., J. Chem. Soc.,96-102 (1951)) (1.24 g, 6.58 mmol) was added to a solution of sodium(0.21 g, 9.13 mmol) in benzyl alcohol (14 mL) under argon. The solutionwas heated in a 135° C. oil bath for 3.5 h, and was poured into water(70 mL) with constant stirring for 10 min. After neutralization withglacial acetic acid, a yellow precipitate formed which was collected byfiltration and washed with water. This solid was crystallized frombenzene to give 5b as a bright yellow crystalline solid: yield, 0.57 g(33%); mp 159°-160° C.; UV (pH 1) λ_(max) 268 nm (ε=0.783×10⁴), 345 (sh)(0.10⁴×10⁴); (pH 6.9) 282 (0.564×10⁴), 345 (sh) (0.338×10⁴); (pH 13) 282(0.549×10⁴), 345 (sh) (0.332×10⁴); ¹ H NMR δ2.35 (s, 3H, CH₃), 5.44 (s,2 H, ArCH₂), 7.34-7.46 (m, 5H, ArH), 7.64 (b s, 2H, NH₂, exchange withD₂ O); MS (EI) calcd. m/z for C₁₂ H₁₂ N₄ O₃ 260.0908, found 260.0913;Anal.(C₁₂ H₁₂ N₄ O₃) C, H, N.

EXAMPLE 21 2,4-Diamino-6-benzyloxy-s-triazine (6)

2,4-Diamino-6-chloro-s-triazine (2.25 g, 15.0 mmol) was added to asolution of sodium (0.43 g, 18.8 mmol) in benzyl alcohol (30 mL) underargon. The suspension was heated in a 130° C. oil bath for 3.5 h. Theexcess benzyl alcohol was removed under vacuum and the resulting solidwas collected with the aid of benzene, and washed with water (100 mL):yield, 1.83 g (56%); mp 184°-185° C. (lit. 186°-188° C.; Wakabayashi etal., Nippon Dojo-Hiryogaku Zasshi, 41, 193-200 (1970)); UV (pH 1)λ_(max) 233 nm (sh) (ε=0.589×10⁴); (pH 6.9) 238 (sh) (0.111×10⁴); (pH13) 240 (sh) (0.073×10⁴); ¹ H NMR δ5.25 (s, 2H, ArCH₂), 6.63 (s, 4H,NH₂, exchange with D₂ O), 7.30-7.42 (m, 5H, ArH); MS (EI) calcd. m/z forC₁₀ H₁₁ N₅ O 217.0963, found 217.0955.

EXAMPLE 22 2-Amino-6-chloro-8-trifluoromethylpurine

A suspension of 8-trifluoromethylguanine (Pfleiderer and Shanshal,Liebigs Ann. Chem., 726, 201-215 (1969)) (2.0 g, 9.1 mmol) inphosphorous oxychloride (20 mL) was refluxed for 3 h. Excess phosphorousoxychloride was evaporated under reduced pressure. The resulting residuewas mixed with ice-water (100 g), and the pH was adjusted to 3-4 with aconcentrated aqueous NaOH solution. The resulting solution was mixedwith MeOH (100 mL) and approximately half (i.e., 100 mL) of the aqueousmethanol solution was loaded on a 3×80 cm Sephadex LH-20 column elutedwith methanol/water (1:1) at 1 mL/min. Column eluent was continuouslymonitored at 280 nm and fractions (10 mL) were collected. The remainderof the reaction mixture in MeOH/H₂ O was chromatographed separatelyunder identical conditions. The desired product eluted in fractions73-85. Evaporation of solvent from the pooled fractions 73-85 from bothchromatographic runs afforded analytically pure2-amino-6-chloro-8-trifluoromethylpurine: yield, 0.94 g (43%); mp >225°C. dec.; UV (pH 1) λ_(max) 245 nm (ε=0.501×10⁴), 314 (0.746×10⁴); (pH6.9) 270 (0.265×10⁴), 315 (0.612×10⁴); (pH 13) 272 (0.269×10⁴), 314(0.612×10⁴); ¹ H NMR δ7.19 (s, 2H, NH₂, exchange with D₂ O), 14.25 (brs, 1H, NH, exchanges with D₂ O); MS (EI) calcd. m/z for C₆ H₃ N₅ F₃ ³⁵Cl237.0029, found 237.0011; calcd. m/z for C₆ H₃ N F ³⁷ Cl 239.0000,found 238.9987; Anal. (C₆ H₃ N₅ F₃ C) C, H, N, F, Cl.

EXAMPLE 23 O⁶ -Benzyl-8-trifluoromethylguanine (1e)

Sodium (0.10 g, 4.3 mmol) was stirred in 5 mL of benzyl alcohol untilall had reacted. 2-Amino-6-chloro-8-trifluoromethylpurine (0.475 g, 2.0mmol) was added, and the reaction mixture was heated in a 135° C. oilbath for 3.5 h. The benzyl alcohol was removed by vacuum distillationyielding a brown oil. The oil was dissolved in water (50 mL) and wasacidified with glacial acetic acid to produce a pale yellow precipitate.The precipitate was collected by filtration and washed with water. Thecrude product was loaded on a 2.5×35 cm silica gel column (Davisil grade633, 200-425 mesh, 60 Å). Elution was carried out with 5% EtOH in CHCl₃to provide analytically pure O⁶ -benzyl-8-trifluoromethylguanine (1e):yield, 0.42 g (67%); mp 214°-216° C. dec.; UV (pH 1) λ_(max) 291 nm(ε=1.229×10⁴); (pH 6.9) 244 (0.470×10⁴), 289 (1.023×10⁴) (pH 13) 247(sh) (0.393×10⁴), 290 (0.923×10⁴); ¹ H NMR δ5.51 (s, 2H, ArCH₂), 6.82(s, 2H, NH₂, exchange with D₂ 0), 7.38-7.55 (m, 5H, ArH), 13.75 (br s,1H, NH, exchanges with D₂ O); MS (EI) calcd. m/z for C₁₃ H₁₀ N₅ OF₃309.0837, found 309.0827; Anal. (Cl₃ H₁₀ ₅ OF₃) C, H, N, F.

EXAMPLE 24 O⁶ -Benzyl-8-trifluoromethyl-9-methylguanine (7)

To O⁶ -benzyl-8-trifluoromethylguanine (1e) (200 mg, 0.65 mmol) underargon was added 0.66 mL of a 1.0M solution of sodium ethoxide inethanol. The solution was stirred for 10 min and the ethanol was removedunder vacuum. The remaining solid was dissolved in anhydrous DMF (1.5mL), and methyl iodide (49 μL, 0.78 mmol) was added to the solution.This solution was stirred at room temperature for 1 h, and 1.5 mLadditional DMF was added. The solution was stirred at room temperatureovernight. The solvent was evaporated under reduced pressure. The crudesolid was loaded on a 2.5×35 cm silica gel column (Davisil grade 633,200-425 mesh, 60 Å). Elution was carried out with chloroform/hexane(3:1) to provide analytically pure O⁶-benzyl-8-trifluoromethyl-9-methylguanine (7): yield, 95 mg (45%); mp86°-89° C.; UV (pH 1) λ_(max) 244 nm (ε=0.581×10⁴), 286 (1.274×10⁴); (pH6.9) 252 (0.608×10⁴), 288 (1.022×10⁴); (pH 13) 252 (0.618×10⁴), 288(1.038×10⁴); ¹ H NMR δ3.70 (s, 3H, CH₃), 5.51 (s, 2H, ArCH₂), 6.91 (s,2H, NH₂, exchange with D₂ 0), 7.38-7.54 (m, 5H, ArH); MS (EI) calcd. m/zfor C₁₄ H₁₂ N5OF₃ 323.0994, found 323.0978; Anal. (C₁₄ H₁₂ N₅ OF₃) C, H,N, F.

All of the references cited herein, including patents, patentapplications, and publications, are hereby incorporated in theirentireties by reference.

While this invention has been described with an emphasis upon preferredembodiments, it will be obvious to those of ordinary skill in the artthat variations of the preferred embodiments may be used and that it isintended that the invention may be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications encompassed within the spirit and scope of the inventionas defined by the following claims.

What is claimed is:
 1. A compound of the formula ##STR8## wherein R₁ isNO₂ and R₂ is a substituent selected from the group consisting ofhydrogen, halo, C₁ -C₄ alkyl, C₁ -C₄ hydroxyalkyl, thiol, C₁ -C₄alkylthio, trifluoromethoxy, oxymethanesulfonyl,oxytrifluoromethanesulfonyl, C₁ -C₄ oxyacyl, C₁ -C₄ aminoalkyl,trifluoromethylamino, ditrifluoromethylamino, aminomethanesulfonyl, C₁-C₄ aminoacyl, aminotrifluoromethylcarbonyl, nitro, nitroso, C₁ -C₄alkyldiazo, C₅ -C₆ aryldiazo, trifluoromethyl, halomethyl, C₁ -C₄haloalkyl, cyanomethyl, C₁ -C₄ cyanoalkyl, cyano, C₁ -C₄alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl, phenyl, phenylcarbonyl, C₁ -C₄acyl, formyl, C₁ -C₄ alkoxymethyl, phenoxymethyl, C₁ -C₄ vinyl, C₁ -C₄ethynyl, and So_(n) R' wherein n is 0, 1, 2, or 3 and R' is hydrogen, C₁-C₄ alkyl, amino, or phenyl.
 2. The compound of claim 16, wherein R₂ ishydrogen.
 3. The compound of claim 1, wherein R₂ is methyl.
 4. Apharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of claim
 1. 5. The pharmaceutical composition ofclaim 4, wherein said pharmaceutically acceptable carrier comprisespolyethylene glycol
 400. 6. A pharmaceutical composition comprising apharmaceutically acceptable carrier and a compound of the formula##STR9## wherein R is a substituent selected from the group consistingof hydrogen, halo, C₁ -C₄ alkyl, C₁ -C₄ hydroxyalkyl, thiol, C₁ -C₄alkylthio, trifluoromethylthio, C₁ -C₄ thioacyl, hydroxy, C₁ -C₄ alkoxy,trifluoromethoxy, oxymethanesulfonyl, oxytrifluoromethanesulfonyl, C₁-C₄ oxyacyl, amino, C₁ -C₄ aminoalkyl, C₁ -C₄ alkylamino, C₁ -C₄dialkylamino, trifluoromethylamino, ditrifluoromethylamino,aminomethanesulfonyl, C₁ -C₄ aminoacyl, aminotrifluoromethylcarbonyl,formylamino, nitro, nitroso, C₁ -C₄ alkyldiazo, C₅ -C₆ aryldiazo,trifluoromethyl, halomethyl, C₁ -C₄ haloalkyl, cyanomethyl, C₁ -C₄cyanoalkyl, cyano, C₁ -C₄ alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl, phenyl,phenylcarbonyl, C₁ -C₄ acyl, formyl, C₁ -C₄ alkoxymethyl, phenoxymethyl,C₁ -C₄ vinyl, C₁ -C₄ ethynyl, and SO_(n) R, wherein n is 0, 1, 2, or 3and R₁ is hydrogen, C₁ -C₄ alkyl, amino, or phenyl, and wherein saidpharmaceutically acceptable carrier comprises polyethylene glycol 400.7. A method of enhancing the chemotherapeutic treatment of tumor cellsin a mammal with an antineoplastic alkylating agent which causescytotoxic lesions at the O⁶ -position of guanine, which methodcomprises:administering to a mammal an effective amount of a compound ofthe formula ##STR10## wherein R₁ is NO₂ or NO, and R₂ is a substituentselected from the group consisting of hydrogen, halo, C₁ -C₄ alkyl, C₁-C₄ hydroxyalkyl, thiol, C₁ -C₄ alkylthio, trifluoromethoxy,oxymethanesulfonyl, oxytrifluoromethanesulfonyl, C₁ -C₄ oxyacyl, C_(l)-C₄ aminoalkyl, trifluoromethylamino, ditrifluoromethylamino,aminomethanesulfonyl, C₁ -C₄ aminoacyl, aminotrifluoromethylcarbonyl,formylamino, nitro, nitroso, C₁ -C₄ alkyldiazo, C₁ -C₆ aryldiazo,trifluoromethyl, halomethyl, C₁ -C₄ haloalkyl, cyanomethyl, C₁ -C₄cyanoalkyl, cyano, C₁ -C₄ alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl, phenyl,phenylcarbonyl, C₁ -C₄ acyl, formyl, C₁ -C₄ alkoxymethyl, phenoxymethyl,C₁ -C₄ vinyl, C₁ -C₄ ethynyl, and SO_(n) R' wherein n is 0, 1, 2, or 3and R' is hydrogen, C_(l) -C₄ alkyl, amino, or phenyl, and administeringto said mammal an effective amount of an antineoplastic alkylating agentwhich causes cytotoxic lesions at the O⁶ -position of guanine.
 8. Amethod of enhancing the chemotherapeutic treatment of tumor cells in amammal with an antineoplastic alkylating agent that causes cytotoxiclesions at the O⁶ -position of guanine, which methodcomprises:administering to a mammal an effective amount of a compound ofthe formula ##STR11## wherein R is a substituent selected from the groupconsisting of hydrogen, halo, C₁ -C₄ alkyl, C₁ -C₄ hydroxyalkyl, thiol,C₁ -C₄ alkylthio, trifluoromethylthio, C₁ -C₄ thioacyl, hydroxy, C₁ -C₄alkoxy, trifluoromethoxy, oxymethanesulfonyl,oxytrifluoromethanesulfonyl, C₁ -C₄ oxyacyl, amino, C₁ -C₄ aminoalkyl,C₁ -C₄ alkylamino, C₁ -C₄ dialkylamino, trifluoromethylamino,ditrifluoromethylamino, aminomethanesulfonyl, C₁ -C₄ aminoacyl,aminotrifluoromethylcarbonyl, formylamino, nitro, nitroso, C₁ -C₄alkyldiazo, C₅ -C₆ aryldiazo, trifluoromethyl, halomethyl, C₁ -C₄haloalkyl, cyanomethyl, C₁ -C₄ cyanoalkyl, cyano, C₁ -C₄alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl, phenyl, phenylcarbonyl, C₁ -C₄acyl, formyl, C₁ -C₄ alkoxymethyl, phenoxymethyl, C₁ -C₄ vinyl, C₁ -C₄ethynyl, and SO_(n) R, wherein n is 0, 1, 2, or 3 and R₁ is hydrogen, C₁-C₄ alkyl, amino, or phenyl, and administering to said mammal aneffective amount of an antineoplastic alkylating agent which causescytotoxic lesions at the O⁶ -position of guanine.
 9. A method ofenhancing the chemotherapeutic treatment of tumor cells in a mammal withan antineoplastic alkylating agent which causes cytotoxic lesions at theO⁶ -position of guanine, which method comprises:administering to amammal an effective amount of a compound of claim 2, and administeringto said mammal an effective amount of an antineoplastic alkylating agentwhich causes cytotoxic lesions at the O⁶ -position of guanine.
 10. Amethod of enhancing the chemotherapeutic treatment of tumor cells in amammal with an antineoplastic alkylating agent which causes cytotoxiclesions at the O⁶ -position of guanine, which methodcomprises:administering to a mammal an effective amount of a compound ofclaim 3, and administering to said mammal an effective amount of anantineoplastic alkylating agent which causes cytotoxic lesions at the O⁶-position of guanine.
 11. A compound of the formula ##STR12## wherein R₁is NO and R₂ is a substituent selected from the group consisting ofhydrogen, halo, C₁ -C₄ alkyl, C_(1-C) ₄ hydroxyalkyl, thiol, C_(1-C) ₄alkylthio, trifluoromethoxy, oxymethanesulfonyl,oxytrifluoromethanesulfonyl, C₁ -C₄ oxyacyl, C₁ -C₄ aminoalkyl,trifluoromethylamino, ditrifluoromethylamino, aminomethanesulfonyl,aminotrifluoromethylcarbonyl, nitro, nitroso, C₁ -C₄ alkyldiazo, C₅ -C₆aryldiazo, trifluoromethyl, halomethyl, C₁ -C₄ haloalkyl, cyanomethyl,C₁ -C₄ cyanoalkyl, cyano, C₁ -C₄ alkyoxycarbonyl, C₁ -C₄ alkylcarbonyl,phenyl, phenylcarbonyl, C₁ -C₄ acyl, formyl, C₁ -C₄ alkoxymethyl,phenoxymethyl, C₁ -C₄ vinyl, C₁ -C₄ ethynyl, and So_(n) R' wherein n is0, 1, 2, or 3 and R' is hydrogen, C₁ -C₄ alkyl, amino, or phenyl. 12.The compound of claim 11, wherein R₂ is hydrogen.
 13. The compound ofclaim 11, wherein R₂ is methyl.
 14. A pharmaceutical compositioncomprising a pharmaceutically acceptable carrier and a compound of claim11.
 15. The pharmaceutical composition of claim 14, wherein saidpharmaceutically acceptable carrier comprises polyethylene glycol 400.16. A method of enhancing the chemotherapeutic treatment of tumor cellsin a mammal with an antineoplastic alkylating agent which causescytotoxic lesions at the O⁶ -position of guanine, which methodcomprises:administering to a mammal an effective amount of a compound ofclaim 11, and administering to said mammal an effective amount of anantineoplastic alkylating agent which causes cytotoxic lesions at the O⁶-position of guanine.
 17. A method of enhancing the chemotherapeutictreatment of tumor cells in a mammal with an antineoplastic alkylatingagent which causes cytotoxic lesions at the O⁶ -position of guanine,which method comprises:administering to a mammal an effective amount ofa compound of claim 12, and administering to said mammal an effectiveamount of an antineoplastic alkylating agent which causes cytotoxiclesions at the O⁶ -position of guanine.
 18. A method of enhancing thechemotherapeutic treatment of tumor cells in a mammal with anantineoplastic alkylating agent which causes cytotoxic lesions at the O⁶-position of guanine, which method comprises:administering to a mammalan effective amount of a compound of claim 13, and administering to saidmammal an effective amount of an antineoplastic alkylating agent whichcauses cytotoxic lesions at the O⁶ -position of guanine.